Ethylenedicysteine (EC)-drug conjugates, compositions and methods for tissue specific disease imaging

ABSTRACT

The invention provides, in a general sense, a new labeling strategy employing  99m Tc chelated with ethylenedicysteine (EC). EC is conjugated with a variety of ligands and chelated to  99m Tc for use as an imaging agent for tissue-specific diseases. The drug conjugates of the invention may also be used as a prognostic tool or as a tool to deliver therapeutics to specific sites within a mammalian body. Kits for use in tissue-specific disease imaging are also provided.

BACKGROUND OF THE INVENTION

The government does not own rights in the present invention.

1. Field of the Invention

The present invention relates generally to the fields of labeling,radioimaging and chemical synthesis. More particularly, it concerns astrategy for radiolabeling target ligands. It further concerns methodsof using those radiolabeled ligands in tumor imaging and tissue-specificdisease imaging.

2. Description of Related Art

Improvement of scintigraphic tumor imaging is extensively determined bydevelopment of more tumor specific radiopharmaceuticals. Due to greatertumor specificity, radiolabeled ligands as well as radiolabeledantibodies have opened a new era in scintigraphic detection of tumorsand undergone extensive preclinical development and evaluation. (Mathiaset al., 1996, 1997a, 1997b). Radionuclide imaging modalities (positronemission tomography, PET; single photon emission computed tomography,SPECT) are diagnostic cross-sectional imaging techniques that map thelocation and concentration of radionuclide-labeled radiotracers.Although CT and MRI provide considerable anatomic information about thelocation and the extent of tumors, these imaging modalities cannotadequately differentiate invasive lesions from edema, radiationnecrosis, grading or gliosis. PET and SPECT can be used to localize andcharacterize tumors by measuring metabolic activity.

The development of new tumor hypoxia agents is clinically desirable fordetecting primary and metastatic lesions as well as predictingradioresponsiveness and time to recurrence. None of the contemporaryimaging modalities accurately measures hypoxia since the diagnosis oftumor hypoxia requires pathologic examination. It is often difficult topredict the outcome of a therapy for hypoxic tumor without knowing atleast the baseline of hypoxia in each tumor treated. Although theEppendorf polarographic oxygen microelectrode can measure the oxygentension in a tumor, this technique is invasive and needs a skillfuloperator. Additionally, this technique can only be used on accessibletumors (e.g., head and neck, cervical) and multiple readings are needed.Therefore, an accurate and easy method of measuring tumor hypoxia willbe useful for patient selection. However, tumor to normal tissue uptakeratios vary depending upon the radiopharmaceuticals used. Therefore, itwould be rational to correlate tumor to normal tissue uptake ratio withthe gold standard Eppendorf electrode measures of hypoxia when newradiopharmaceuticals are introduced to clinical practice.

[¹⁸F]FMISO has been used to diagnose head and neck tumors, myocardialinfarction, inflammation, and brain ischemia (Martin et al. 1992; Yeh etal. 1994; Yeh et al. 1996; Liu et al. 1994). Tumor to normal tissueuptake ratio was used as a baseline to assess tumor hypoxia (Yet et al.1996). Although tumor hypoxia using [¹⁸F]FMISO was clearly demonstrated,introducing new imaging agents into clinical practice depends on someother factors such as easy availability and isotope cost. Although tumormetabolic imaging using [¹⁸F]FDG was clearly demonstrated, introducingmolecular imaging agents into clinical practice depends on some otherfactors such as easy availability and isotope cost.[¹⁸F]fluorodeoxyglucose (FDG) has been used to diagnose tumors,myocardial infarction, and neurological disease. In addition, PETradiosynthesis must be rapid because of short half-life of the positronisotopes. ¹⁸F chemistry is also complex. The ¹⁸F chemistry is notreproducible in different molecules. Thus, it would be ideal to developa chelator which could conjugate to various drugs. The preferred isotopewould be ^(99m)Tc due to low cost ($0.21/mCi vs. $50/mCi for ¹⁸F) andlow energy (140 Kev vs. 571 Kev for ¹⁸F). ^(99m)Tc is easily obtainedfrom a ⁹⁹Mo generator. Due to favorable physical characteristics as wellas extremely low price, ^(99m)Tc has been preferred to labelradiopharmaceuticals.

Several compounds have been labeled with ^(99m)Tc using nitrogen andsulfur chelates (Blondeau et al., 1967; Davison et al., 1980).Bis-aminoethanethiol tetradentate ligands, also called diaminoditholcompounds, are known to form very stable Tc(V)O complexes on the basisof efficient binding of the oxotechnetium group to two thiolsulfur andtwo amine nitrogen atoms. ^(99m)Tc-L,L-ethylenedicysteine (^(99m)Tc-EC)is a recent and successful example of N₂S₂ chelates. EC can be labeledwith ^(99m)Tc easily and efficiently with high radiochemical purity andstability, and is excreted through the kidney by active tubulartransport (Surma et al., 1994; Van Nerom et al., 1990, 1993; Verbruggenet al., 1990, 1992). Other applications of EC would be chelated withgalium-68 (a positron emitter, t½=68 min) for PET and gadolinium, ironor manganese for magnetic resonance imaging (MRI). ^(99m)Tc-EC-neomycinand ^(99m)Tc-EC-deoxyglucose were developed and their potential use intumor characterization was evaluated.

SUMMARY OF THE INVENTION

The present invention overcomes these and other drawbacks of the priorart by providing a new radiolabeling strategy to target tissues forimaging. The invention provides radiolabeled tissue-specific ligands, aswell as methods for making the radiolabeled ligands and for using themto image tissue-specific diseases.

The present invention provides compositions for tissue specific diseaseimaging. The imaging compositions of the invention generally include aradionuclide label chelated with ethylenedicysteine and a tissuespecific ligand conjugated to the ethylenedicysteine on one or both ofits acid arms. The ethylenedicysteine forms an N₂S₂ chelate with theradionuclide label. Of course, the chelated compound will include anionic bond between the ranionuclide and the chelating compound. Theterms “EC-tissue specific ligand conjugate,” “EC-derivative” and“EC-drug conjugate” are used interchangeably herein to refer to theunlabeled ethylenedicysteine-tissue specific ligand compound. As usedherein, the term “conjugate” refers to a covalently bonded compound.

Ethylenedicysteine is a bis-aminoethanethiol (BAT) tetradentate ligand,also known as diaminodithiol (DADT) compounds. Such compounds are knownto form very stable Tc(V)O-complexes on the basis of efficient bindingof the oxotechnetium group to two thiol-sulphur and two amine-nitrogenatoms. The ^(99m)Tc labeled diethylester (^(99m)Tc-L,L-ECD) is known asa brain agent. ^(99m)Tc-L,L-ethylenedicysteine (^(99m)Tc-L,L-EC) is itsmost polar metabolite and was discovered to be excreted rapidly andefficiently in the urine. Thus, ^(99m)Tc-L,L-EC has been used as a renalfunction agent. (Verbruggen et al. 1992).

A tissue specific ligand is a compound that, when introduced into thebody of a mammal or patient, will specifically bind to a specific typeof tissue. It is envisioned that the compositions of the invention mayinclude virtually any known tissue specific compound. Preferably, thetissue specific ligand used in conjunction with the present inventionwill be an anticancer agent, DNA topoisomerase inhibitor,antimetabolite, tumor marker, folate receptor targeting ligand, tumorapoptotic cell targeting ligand, tumor hypoxia targeting ligand, DNAintercalator, receptor marker, peptide, nucleotide, organ specificligand, antimicrobial agent, such as an antibiotic or an antifungal,glutamate pentapeptide or an agent that mimics glucose. The agents thatmimic glucose may also be referred to as “sugars.”

Preferred anticancer agents include methotrexate, doxorubicin,tamoxifen, paclitaxel, topotecan, LHRH, mitomycin C, etoposide, tomudex,podophyllotoxin, mitoxantrone, captothecin, colchicine, endostatin,fludarabin and gemcitabine. Preferred tumor markers include PSA, ER, PR,AFP, CA-125, CA-199, CEA, interferons, BRCA1, cytoxan, p53, VEGF,integrins, endostatin, HER-2/neu, antisense markers or a monoclonalantibody. It is envisioned that any other known tumor marker or anymonoclonal antibody will be effective for use in conjunction with theinvention. Preferred folate receptor targeting ligands include folate,methotrexate and tomudex. Preferred tumor apoptotic cell or tumorhypoxia targeting ligands include annexin V, colchicine, nitroimidazole,mitomycin or metronidazole. Preferred antimicrobials include ampicillin,amoxicillin, penicillin, cephalosporin, clidamycin, gentamycin,kanamycin, neomycin, natamycin, nafcillin, rifampin, tetracyclin,vancomycin, bleomycin, and doxycyclin for gram positive and negativebacteria and amphotericin B, amantadine, nystatin, ketoconazole,polymycin, acyclovir, and ganciclovir for fungi. Preferred agents thatmimic glucose, or sugars, include neomycin, kanamycin, gentamycin,paromycin, amikacin, tobramycin, netilmicin, ribostamycin, sisomicin,micromicin, lividomycin, dibekacin, isepamicin, astromicin,aminoglycosides, glucose or glucosamine.

In certain embodiments, it will be necessary to include a linker betweenthe ethylenedicysteine and the tissue specific ligand. A linker istypically used to increase drug solubility in aqueous solutions as wellas to minimize alteration in the affinity of drugs. While virtually anylinker which will increase the aqueous solubility of the composition isenvisioned for use in conjunction with the present invention, thelinkers will generally be either a poly-amino acid, a water solublepeptide, or a single amino acid. For example, when the functional groupon the tissue specific ligand, or drug, is aliphatic or phenolic-OH,such as for estradiol, topotecan, paclitaxel, or raloxifen etoposide,the linker may be poly-glutamic acid (MW about 750 to about 15,000),poly-aspartic acid (MW about 2,000 to about 15,000), bromo ethylacetate,glutamic acid or aspartic acid. When the drug functional group isaliphatic or aromatic-NH₂ or peptide, such as in doxorubicin, mitomycinC, endostatin, annexin V, LHRH, octreotide, and VIP, the linker may bepoly-glutamic acid (MW about 750 to about 15,000), poly-aspartic acid(MW about 2,000 to about 15,000), glutamic acid or aspartic acid. Whenthe drug functional group is carboxylic acid or peptide, such as inmethotrexate or folic acid, the linker may be ethylenediamine, orlysine.

While the preferred radionuclide for imaging is ^(99m)Tc, it isenvisioned that other radionuclides may be chelated to the EC-tissuespecific ligand conjugates, or EC-drug conjugates of the invention,especially for use as therapeutics. For example, other usefulradionuclides are ¹⁸⁸Re, ¹⁸⁶Re, ¹⁵³Sm, ¹⁶⁶Ho, ⁹⁰Y, ⁸⁹Sr, ⁶⁷Ga, ⁶⁸Ga,¹¹¹In, ¹⁵³Gd, and ⁵⁹Fe. These compositions are useful to deliver thetherapeutic radionuclides to a specific lesion in the body, such asbreast cancer, ovarian cancer, prostate cancer (using for example,^(186/188)Re-EC-folate) and head and neck cancer (using for example,^(186/188)Re-EC-nitroimidazole).

Specific embodiments of the present invention include^(99m)Tc-EC-annexin V, ^(99m)Tc-EC-colchicine,^(99m)Tc-EC-nitroimidazole, ^(99m)Tc-EC-glutamate pentapeptide,^(99m)Tc-EC-metronidazole, ^(99m)Tc-EC-folate, ^(99m)Tc-EC-methotrexate,^(99m)Tc-EC-tomudex, ^(99m)Tc-EC-neomycin, ^(99m)Tc-EC-kanamycin,^(99m)Tc-EC-aminoglycosides, (glucosamine, EC-deoxyglucose),^(99m)Tc-EC-gentamycin, and ^(99m)Tc-EC-tobramycin.

The present invention further provides a method of synthesizing aradiolabeled ethylenedicysteine drug conjugate or derivative for imagingor therapeutic use. The method includes obtaining a tissue specificligand, admixing the ligand with ethylenedicysteine (EC) to obtain anEC-tissue specific ligand derivative, and admixing the EC-tissuespecific ligand derivative with a radionuclide and a reducing agent toobtain a radionuclide labeled EC-tissue specific ligand derivative. Theradionuclide is chelated to the EC via an N₂S₂ chelate. The tissuespecific ligand is conjugated to one or both acid arms of the EC eitherdirectly or through a linker as described above. The reducing agent ispreferably a dithionite ion, a stannous ion or a ferrous ion.

The present invention further provides a method for labeling a tissuespecific ligand for imaging, therapeutic use or for diagnostic orprognostic use. The labeling method includes the steps of obtaining atissue specific ligand, admixing the tissue specific ligand withethylenedicysteine (EC) to obtain an EC-ligand drug conjugate, andreacting the drug conjugate with ^(99m)Tc in the presence of a reducingagent to form an N₂S₂ chelate between the ethylenedicysteine and the^(99m)Tc.

For purposes of this embodiment, the tissue specific ligand may be anyof the ligands described above or discussed herein. The reducing agentmay be any known reducing agent, but will preferably be a dithioniteion, a stannous ion or a ferrous ion.

In another embodiment, the present invention provides a method ofimaging a site within a mammalian body. The imaging method includes thesteps of administering an effective diagnostic amount of a compositioncomprising a ^(99m)Tc labeled ethylenedicysteine-tissue specific ligandconjugate and detecting a radioactive signal from the ^(99m)Tc localizedat the site. The detecting step will typically be performed from about10 minutes to about 4 hours after introduction of the composition intothe mammalian body. Most preferably, the detecting step will beperformed about 1 hour after injection of the composition into themammalian body.

In certain preferred embodiments, the site will be an infection, tumor,heart, lung, brain, liver, spleen, pancreas, intestine or any otherorgan. The tumor or infection may be located anywhere within themammalian body but will generally be in the breast, ovary, prostate,endometrium, lung, brain, or liver. The site may also be afolate-positive cancer or estrogen-positive cancer.

The invention also provides a kit for preparing a radiopharmaceuticalpreparation. The kit generally includes a sealed via or bag, or anyother kind of appropriate container, containing a predetermined quantityof an ethylenedicysteine-tissue specific ligand conjugate compositionand a sufficient amount of reducing agent to label the conjugate with^(99m)Tc. In certain cases, the ethylenedicysteine-tissue specificligand conjugate composition will also include a linker between theethylenedicysteine and the tissue specific ligand. The tissue specificligand may be any ligand that specifically binds to any specific tissuetype, such as those discussed herein. When a linker is included in thecomposition, it may be any linker as described herein.

The components of the kit may be in any appropriate form, such as inliquid, frozen or dry form. In a preferred embodiment, the kitcomponents are provided in lyophilized form. The kit may also include anantioxidant and/or a scavenger. The antioxidant may be any knownantioxidant but is preferably vitamin C. Scavengers may also be presentto bind leftover radionuclide. Most commercially available kits containglucoheptonate as the scavenger. However, glucoheptonate does notcompletely react with typical kit components, leaving approximately10-15% left over. This leftover glucoheptonate will go to a tumor andskew imaging results. Therefore, the inventors prefer to use EDTA as thescavenger as it is cheaper and reacts more completely.

Another aspect of the invention is a prognostic method for determiningthe potential usefulness of a candidate compound for treatment ofspecific tumors. Currently, most tumors are treated with the “usual drugof choice” in chemotherapy without any indication whether the drug isactually effective against that particular tumor until months, and manythousands of dollars, later. The imaging compositions of the inventionare useful in delivering a particular drug to the site of the tumor inthe form of a labeled EC-drug conjugate and then imaging the site withinhours to determine whether a particular drug.

In that regard, the prognostic method of the invention includes thesteps of determining the site of a tumor within a mammalian body,obtaining an imaging composition which includes a radionuclide chelatedto EC which is conjugated to a tumor specific cancer chemotherapy drugcandidate, administering the composition to the mammalian body andimaging the site to determine the effectiveness of the candidate drugagainst the tumor. Typically, the imaging step will be performed withinabout 10 minutes to about 4 hours after injection of the compositioninto the mammalian body. Preferably, the imaging step will be performedwithin about 1 hour after injection of the composition into themammalian body.

The cancer chemotherapy drug candidate to be conjugated to EC in theprognostic compositions may be chosen from known cancer chemotherapydrugs. Such drugs appear in Table 2. There are many anticancer agentsknown to be specific for certain types of cancers. However, not everyanticancer agent for a specific type of cancer is effective in everypatient. Therefore, the present invention provides for the first time amethod of determining possible effectiveness of a candidate drug beforeexpending a lot of time and money on treatment.

Yet another embodiment of the present invention is a reagent forpreparing a scintigraphic imaging agent. The reagent of the inventionincludes a tissue specific ligand, having an affinity for targeted sitesin vivo sufficient to produce a scintigraphically-detectable image,covalently linked to a ^(99m)Tc binding moiety. The ^(99m)Tc bindingmoiety is either directly attached to the tissue specific ligand or isattached to the ligand through a linker as described above. The ^(99m)Tcbinding moiety is preferably an N₂S₂ chelate between ^(99m)Tc in the +4oxidation state and ethylenedicysteine (EC). The tissue specific ligandwill be covalently linked to one or both acid arms of the EC, eitherdirectly or through a linker as described above. The tissue specificligand may be any of the ligands as described above.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. Synthetic scheme of ^(99m)Tc-EC-folate.

FIG. 2. Synthetic scheme of ^(99m)Tc-EC-MTX (methotrexate).

FIG. 3. Synthetic scheme of ^(99m)Tc-EC-TDX (tomudex).

FIG. 4. Biodistribution studies for ^(99m)Tc-EC and ^(99m)Tc-EC-folate.

FIG. 5. Blocking studies for tumor/muscle and tumor/blood count ratioswith ^(99m)Tc-EC-folate.

FIGS. 6A and 6B. Scintigraphic images of tumor in ^(99m)Tc-EC-folateinjected group as compared to ^(99m)Tc-EC injected group.

FIG. 7. Synthetic scheme of EC-MN (metronidazole)

FIG. 8A and FIG. 8B. For EC-NIM, FIG. 8A shows the synthetic scheme andFIG. 8B illustrates the ¹H-NMR confirmation of the structure.

FIG. 9. Biodistribution studies (tumor/blood ratios) for ^(99m)Tc-EC-MN,[¹⁸F]FMISO and [¹³¹I]IMISO.

FIG. 10. Biodistribution studies (tumor/muscle ratios) for ^(99m)Tc-EC,[¹⁸F]FMISO and [¹³¹I]IMISO.

FIGS. 11A and 11B. Scintigraphic images of tumor in ^(99m)Tc-EC-MN (FIG.11A) and ^(99m)Tc-EC (FIG. 11B) injected groups.

FIG. 12. Autoradiograms performed at 1 hour after injection with^(99m)Tc-EC-MN.

FIG. 13. Illustrates stability of ^(99m)Tc-EC-NIM in dog serum samples.

FIG. 14A and FIG. 14B. Illustrates breast tumor uptake of^(99m)Tc-EC-NIM vs. ^(99m)Tc-EC in rats (FIG. 14A) and in rats treatedwith paclitaxel compared to controls (FIG. 14B).

FIG. 15A, FIG. 15B, FIG. 15C, and FIG. 15D. Illustrates ovarian tumoruptake of ^(99m)Tc-EC-NIM vs. ^(99m)Tc-EC in rats (FIG. 15A) The tumoruptake in rats treated with paclitaxel (FIG. 15B) was less than tumoruptake in rats not treated with paclitaxel (FIG. 15A). Also illustratedis tumor uptake of ^(99m)Tc-EC-NIM in rats having sarcomas. FIG. 15Cshows tumor uptake in sarcoma bearing rats treated with paclitaxel whileFIG. 15D shows tumor uptake in rats not treated with paclitaxel. Therewas a decreased uptake of ^(99m)Tc-EC-NIM after treatment withpaclitaxel.

FIG. 16. Synthetic scheme of EC-GAP (pentaglutamate).

FIG. 17. Scintigraphic images of breast tumors in ^(99m)Tc-EC-GAPinjected group.

FIG. 18. Scintigraphic images of breast tumors in ^(99m)Tc-EC-ANNEX Vinjected group at different time intervals.

FIG. 19A and FIG. 19B. Comparison of uptake difference of^(99m)Tc-EC-ANNEX V between pre-(FIG. 19A) and post-(FIG. 19B)paclitaxel treatment in ovarian tumor bearing group.

FIG. 20A and FIG. 20B. Comparison of uptake difference of^(99m)Tc-EC-ANNEX V between pre-(FIG. 20A) and post-(FIG. 20B)paclitaxel treatment in sarcoma tumor bearing group.

FIG. 21. Synthetic scheme of EC-COL (-colchicine).

FIG. 22. Illustration that no degradation products observed in EC-COLsynthesis.

FIG. 23. Ratios of tumor to muscle and tumor to blood as function oftime for ^(99m)Tc-EC-COL.

FIG. 24. Ratios of tumor to muscle and tumor to blood as function oftime for ^(99m)Tc-EC.

FIG. 25. In vivo imaging studies in breast tumor bearing rats with^(99m)Tc-EC-COL.

FIG. 26. In vivo imaging studies in breast tumor bearing rats with^(99m)Tc-EC.

FIG. 27. Computer outlined region of interest after injection of^(99m)Tc-EC-COL vs. ^(99m)Tc-EC.

FIG. 28. SPECT with ^(99m)Tc-EC-MN of 59 year old male patient whosuffered stroke. Images taken one hour post-injection.

FIG. 29. MRI T1 weighted image of same patient as FIG. 28.

FIG. 30. SPECT with ^(99m)Tc-EC-MN of 73 year old male patient one dayafter stroke at one hour post-injection.

FIG. 31. SPECT with ^(99m)Tc-EC-MN of same 73 year old patient as imagedin FIG. 30 twelve days after stroke at one hour post-injection.

FIG. 32. CT of same 73 year old male stroke patient as imaged in FIG.30, one day after stroke.

FIG. 33. CT of same 73 year old male stroke patient as imaged in FIG.32, twelve days after stroke. Note, no marked difference between daysone and twelve using CT for imaging.

FIG. 34. SPECT with 99mTc-EC-MN of 72 year old male patient who suffereda stroke at one hour post-injection.

FIG. 35. CT of same 72 year old stroke patient as imaged in FIG. 34.Note how CT image exaggerates the lesion size.

FIG. 36. Synthetic scheme of 99mTc-EC-neomycin.

FIG. 37A. Scintigraphic image of breast tumor-bearing rats afteradministration of ^(99m)Tc-EC and ^(99m)Tc-EC-neomycin (100 μCi/rat,iv.) showed that the tumor could be well visualized from 0.5-4 hourspostinjection.

FIG. 37B. Scintimammography with ^(99m)Tc-EC-neomycin (30 mCi, iv.) of abreast cancer patient. Images taken two hours post-injection.

FIG. 38A. ¹H-NMR of EC.

FIG. 38B. ¹H-NMR of neomycin.

FIG. 38C. ¹H-NMR of EC-neomycin.

FIG. 39A and FIG. 39B. Mass spectrometry of EC-neomycin (M+1112.55).

FIG. 40A. UV wavelength scan of EC.

FIG. 40B. UV wavelength scan of neomycin.

FIG. 40C. UV wavelength scan of EC-neomycin.

FIG. 41. Radio-TLC analysis of ^(99m)Tc-EC-neomycin.

FIG. 42. HPLC analysis of ^(99m)Tc-EC-neomycin (radioactive detector).

FIG. 43. HPLC analysis of ^(99m)Tc-EC-neomycin (UV 254 nm).

FIG. 44. HPLC analysis of ¹⁸F-FDG (radioactive detector).

FIG. 45. HPLC analysis of ¹⁸F-FDG (UV 254 nm).

FIG. 46. In vitro cellular uptake assay of a series of ^(99m)Tc-EC-drugconjugates in lung cancer cell line. ^(99m)Tc-EC-neomycin showed highestuptake in the agents tested.

FIG. 47. Effect of glucose on cellular (A549) uptake of^(99m)Tc-EC-neomycin and ¹⁸F-FDG.

FIG. 48A and FIG. 48B. Effect of glucose on cellular (H1299) uptake of^(99m)Tc-EC-neomycin and ¹⁸F-FDG illustrated as percent of drug uptake(FIG. 48A) and as percent of change with glucose loading (FIG. 48B).

FIG. 49. Synthetic scheme of ^(99m)Tc-EC-Glucosamine

FIG. 50. Hexokinase assay of glucose.

FIG. 51. Hexokinase assay of glucosamine.

FIG. 52. Hexokinase assay of EC-glucosamine.

FIG. 53. Hexokinase assay of EC-GAP-glucosamine.

FIG. 54. Synthetic scheme of ^(99m)Tc-EC-GAP-glucosamine.

FIG. 55A, FIG. 55B, FIG. 55C. In vitro cellular uptake assay of^(99m)Tc-EC (FIG. 56A), ^(99m)Tc-EC-deoxyglucose-GAP (FIG. 56B), and¹⁸F-FDG (FIG. 56C) in lung cancer cell line (A549). ^(99m)Tc-EC-DGshowed similar uptake compared to ¹⁸F-FDG.

FIG. 56. Tumor-to-tissue count density ratios of ^(99m)Tc-EC-GAP inbreast tumor-bearing rats.

FIG. 57 In vitro cellular uptake of ¹⁸PDG with glucose loading at 2hours post-injection in breast cancer cell line (13762).

FIG. 58. In vivo tissue uptake of ^(99m)Tc-EC-neomycin in breasttumor-bearing mice.

FIG. 59. Synthetic scheme of ^(99m)Tc-EC-deoxyglucose.

FIG. 60. Mass spectrometry of EC-deoxyglucose.

FIG. 61. ¹H-NMR of EC-deoxyglucose (EC-DG).

FIG. 62. ¹H-NMR of glucosamine.

FIG. 63. Radio-TLC analysis of ^(99m)Tc-EC-DG.

FIG. 64. HPLC analysis of ^(99m)Tc-EC-deoxyglucose and^(99m)Tc-EC-(radioactive detector).

FIG. 65. HPLC analysis of ^(99m)Tc-EC-deoxyglucose and ^(99m)Tc-EC(radioactive detector, mixed).

FIG. 66. Hexokinase assay of glucose.

FIG. 67. Hexokinase assay of FDG.

FIG. 68. Hexokinase assay of EC-DG.

FIG. 69. In vitro cellular uptake assay of ^(99m)Tc-EC-deoxyglucose,^(99m)Tc-EC and ¹⁸F-FDG in lung cancer cell line (A549). ^(99m)Tc-EC-DGshowed similar uptake compared to ¹⁸F-FDG.

FIG. 70. Effect of d- and 1-glucose on breast cellular (13762 cell line)uptake of ^(99m)Tc-EC-DG.

FIG. 71. Effect of d- and 1-glucose on breast cellular (13762 cell line)uptake of ¹⁸F-FDG.

FIG. 72. Effect of d- and 1-glucose on lung cellular (A549 cell line)uptake of ¹⁸F-FDG.

FIG. 73. Effect of d- and 1-glucose on breast cellular (A549 cell line)uptake of ^(99m)Tc-EC-DG.

FIG. 74. Effect of in vivo blood glucose level induced by glucosamineand EC-DG (1.2 mmol/kg, i.v.).

FIG. 75. Effect of in vivo blood glucose level induced by FDG (1.2 and1.9 mmol/kg, i.v.) and insulin.

FIG. 76. Tumor-to-tissue count density ratios of^(99m)Tc-EC-deoxyglucose in breast tumor-bearing rats.

FIG. 77. In vivo biodistribution of ^(99m)Tc-EC-deoxyglucose in breasttumor-bearing rats.

FIG. 78. In vivo tissue uptake of ^(99m)Tc-EC-deoxyglucose in lungtumor-bearing mice.

FIG. 79. In vivo tissue uptake of ^(99m)Tc-EC-neomycin in lungtumor-bearing mice.

FIG. 80. In vivo tissue uptake of ¹⁸F-FDG in lung tumor-bearing mice.

FIG. 81. Planar image of breast tumor-bearing rats after administrationof ^(99m)Tc-EC and ^(99m)Tc-EC-deoxyglucose (100 μCi/rat, iv.) showedthat the tumor could be well visualized from 0.5-4 hours postinjection.

FIG. 82A. MRI of a patient with malignant astrocytoma.

FIG. 82B. SPECT with ^(99m)Tc-EC-DG of a patient with malignantastrocytoma.

FIG. 83A. MRI of a patient with hemorrhagic astrocytoma.

FIG. 83B. SPECT with ^(99m)Tc-EC-DG of a patient with malignantastrocytoma.

FIG. 84A. MRI of a patient with benign meningioma.

FIG. 84B. SPECT with ^(99m)Tc-EC-DG of a patient with benign meningiomashowed no focal intensed uptake.

FIG. 85A. CT of a patient with TB in lung.

FIG. 85B. SPECT with ^(99m)Tc-EC-DG of a patient with TB showed no focalintensed uptake.

FIG. 86A. CT of patient with lung cancer.

FIG. 86B. Whole body images of ^(99m)Tc-EC-DG of a patient with lungcancer.

FIG. 86C. SPECT with ^(99m)Tc-EC-DG of a patient with lung cancer, thetumor showed focal intensed uptake.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

In the field of nuclear medicine, certain pathological conditions arelocalized, or their extent is assessed, by detecting the distribution ofsmall quantities of internally-administered radioactively labeled tracercompounds (called radiotracers or radiopharmaceuticals). Methods fordetecting these radiopharmaceuticals are known generally as imaging orradioimaging methods.

In radioimaging, the radiolabel is a gamma-radiation emittingradionuclide and the radiotracer is located using a gamma-radiationdetecting camera (this process is often referred to as gammascintigraphy). The imaged site is detectable because the radiotracer ischosen either to localize at a pathological site (termed positivecontrast) or, alternatively, the radiotracer is chosen specifically notto localize at such pathological sites (termed negative contrast).

A variety of radionuclides are known to be useful for radioimaging,including ⁶⁷Ga, ^(99m)Tc, ¹¹¹In, ¹²³I, ¹²⁵I, ¹⁶⁹Yb or ¹⁸⁶Re. Due tobetter imaging characteristics and lower price, attempts have been madeto replace the ¹²³I, ¹³¹I, ⁶⁷Ga and ¹¹¹In labeled compounds withcorresponding ^(99m)Tc labeled compounds when possible. Due to favorablephysical characteristics as well as extremely low price ($0.21/mCi),^(99m)Tc has been preferred to label radiopharmaceuticals. Although ithas been reported that DTPA-drug conjugate could be labeled with^(99m)Tc effectively (Mathias et al., 1997), DTPA moiety does notchelate with ^(99m)Tc as stable as with ¹¹¹In. (Goldsmith, 1997).

A number of factors must be considered for optimal radioimaging inhumans. To maximize the efficiency of detection, a radionuclide thatemits gamma energy in the 100 to 200 keV range is preferred. To minimizethe absorbed radiation dose to the patient, the physical half-life ofthe radionuclide should be as short as the imaging procedure will allow.To allow for examinations to be performed on any day and at any time ofthe day, it is advantageous to have a source of the radionuclide alwaysavailable at the clinical site. ^(99m)Tc is a preferred radionuclidebecause it emits gamma radiation at 140 keV, it has a physical half-lifeof 6 hours, and it is readily available on-site using amolybdenum-99/technetium-99m generator.

Bis-aminoethanethiol tetradentate ligands, also called diaminodithiolcompounds, are known to form very stable Tc(V)O-complexes on the basisof efficient binding of the oxotechnetium group to two thiolsulfur andtwo amine nitrogen atoms. (Davison et al., 1980; 1981; Verbruggen etal., 1992). ^(99m)Tc-L,L-ethylenedicysteine (^(99m)Tc-EC) is the mostrecent and successful example of N₂S₂ chelates. (Verbruggen et al.,1992; Van Nerom et al., 1993; Surma et al., 1994). EC, a new renalimaging agent, can be labeled with ^(99m)Tc easily and efficiently withhigh radiochemical purity and stability and is excreted through kidneyby active tubular transport. (Verbruggen et al., 1992; Van Nerom et al.,1993; Surma et al., 1994; Verbruggen et al., 1990; Van Nerom et al.,1990; Jamar et al., 1993). Other applications of EC would be chelatedwith galium-68 (a positron emitter, t½=68 minutes) for PET andgadolinium, iron or manganese for magnetic resonance imaging (MRI).

The present invention utilizes ^(99m)Tc-EC as a labeling agent to targetligands to specific tissue types for imaging. The advantage ofconjugating the EC with tissue targeting ligands is that the specificbinding properties of the tissue targeting ligand concentrates theradioactive signal over the area of interest. While it is envisionedthat the use of ^(99m)Tc-EC as a labeling strategy can be effective withvirtually any type of compound, some suggested preferred ligands areprovided herein for illustration purposes. It is contemplated that the^(99m)Tc-EC-drug conjugates of the invention may be useful to image notonly tumors, but also other tissue-specific conditions, such asinfection, hypoxic tissue (stroke), myocardial infarction, apoptoticcells, Alzheimer's disease and endometriosis.

Radiolabeled proteins and peptides have been reported in the prior art.(Ege et al., U.S. Pat. No. 4,832,940, Abrams et al., 1990; Bakker etal., 1990; Goldsmith et al., 1995, 1997; Olexa et al. 1982; Ranby et al.1988; Hadley et al. 1988; Lees et al. 1989; Sobel et al. 1989; Stuttle,1990; Maraganore et al. 1991; Rodwell et al. 1991; Tubis et al. 1968;Sandrehagen 1983). However, ^(99m)Tc-EC has not been used in conjunctionwith any ligands, other than as the diethylester (Kabasakal, 2000),prior to the present invention. The diethylester of EC was used as acerebral blood flow agent (Kikukawa, et al., 2000).

Although optimal for radioimaging, the chemistry of ^(99m)Tc has notbeen as thoroughly studied as the chemistry of other elements and forthis reason methods of radiolabeling with ^(99m)Tc are not abundant.^(99m)Tc is normally obtained as ^(99m)Tc pertechnetate (TcO4⁻;technetium in the +7 oxidation state), usually from amolybdenum-99/technetium-99m generator. However, pertechnetate does notbind well with other compounds. Therefore, in order to radiolabel acompound, ^(99m)Tc pertechnetate must be converted to another form.Since technetium does not form a stable ion in aqueous solution, it mustbe held in such solutions in the form of a coordination complex that hassufficient kinetic and thermodynamic stability to prevent decompositionand resulting conversion of ^(99m)Tc either to insoluble technetiumdioxide or back to pertechnetate.

For the purpose of radiolabeling, it is particularly advantageous forthe ^(99m)Tc complex to be formed as a chelate in which all of the donorgroups surrounding the technetium ion are provided by a single chelatingligand—in this case, ethylenedicysteine. This allows the chelated^(99m)Tc to be covalently bound to a tissue specific ligand eitherdirectly or through a single linker between the ethylenedicysteine andthe ligand.

Technetium has a number of oxidation states: +1, +2, +4, +5, +6 and +7.When it is in the +1 oxidation state, it is called Tc MIBI. Tc MIBI mustbe produced with a heat reaction. (Seabold et al. 1999). For purposes ofthe present invention, it is important that the Tc be in the +4oxidation state. This oxidation state is ideal for forming the N₂S₂chelate with EC. Thus, in forming a complex of radioactive technetiumwith the drug conjugates of the invention, the technetium complex,preferably a salt of ^(99m)Tc pertechnetate, is reacted with the drugconjugates of the invention in the presence of a reducing agent.

The preferred reducing agent for use in the present invention isstannous ion in the form of stannous chloride (SnCl₂) to reduce the Tcto its +4 oxidation state. However, it is contemplated that otherreducing agents, such as dithionate ion or ferrous ion may be useful inconjunction with the present invention. It is also contemplated that thereducing agent may be a solid phase reducing agent. The amount ofreducing agent can be important as it is necessary to avoid theformation of a colloid. It is preferable, for example, to use from about10 to about 100 μg SnCl₂ per about 100 to about 300 mCi of Tcpertechnetate. The most preferred amount is about 0.1 mg SnCl₂ per about200 mCi of Tc pertechnetate and about 2 ml saline. This typicallyproduces enough Tc-EC-tissue specific ligand conjugate for use in 5patients.

It is often also important to include an antioxidant in the compositionto prevent oxidation of the ethylenedicysteine. The preferredantioxidant for use in conjunction with the present invention is vitaminC (ascorbic acid). However, it is contemplated that other antioxidants,such as tocopherol, pyridoxine, thiamine or rutin, may also be useful.

In general, the ligands for use in conjunction with the presentinvention will possess either amino or hydroxy groups that are able toconjugate to EC on either one or both acid arms. If amino or hydroxygroups are not available (e.g., acid functional group), a desired ligandmay still be conjugated to EC and labeled with ^(99m)Tc using themethods of the invention by adding a linker, such as ethylenediamine,amino propanol, diethylenetriamine, aspartic acid, polyaspartic acid,glutamic acid, polyglutamic acid, or lysine. Ligands contemplated foruse in the present invention include, but are not limited to,angiogenesis/antiangiogenesis ligands, DNA topoisomerase inhibitors,glycolysis markers, antimetabolite ligands, apoptosis/hypoxia ligands,DNA intercalators, receptor markers, peptides, nucleotides,antimicrobials such as antibiotics or antifungals, organ specificligands and sugars or agents that mimic glucose.

EC itself is water soluble. It is necessary that the EC-drug conjugateof the invention also be water soluble. Many of the ligands used inconjunction with the present invention will be water soluble, or willform a water soluble compound when conjugated to EC. If the tissuespecific ligand is not water soluble, however, a linker which willincrease the solubility of the ligand may be used. Linkers may attach toan aliphatic or aromatic alcohol, amine or peptide or to a carboxylicand or peptide. Linkers may be either poly amino acid (peptide) or aminoacid such as glutamic acid, aspartic acid or lysine. Table I illustratesdesired linkers for specific drug functional groups. TABLE 1 DrugFunctional Group Linker Example Aliphatic or phenolio-OH EC-Poly(glutamic acid) A (MW. 750-15,000) or EC. poly(aspertic acid) (MW.2000-15,000) or bromo ethylacetate or EC-glutamic acid or EC-asperticacid. Aliphatic or aromatic-NH₂ EC-poly(glutamic acid) B or peptide (MW.750-15,000) or EC- poly(aspertic acid) (MW. 2000-15,000) or EC- glutamicacid (mono- or diester) or EC-aspartic acid. Carboxylic acid or peptideEthylene diamine, lysine CExamples:A. estradiol, topotecan, paclitaxel, raloxlfen etoposideB. doxorubicin, mitomycin C, endostatin, annexin V. LHRH, octreotide,VIPC. methotrexate, folic acid

It is also envisioned that the EC-tissue specific ligand drug conjugatesof the invention may be chelated to other radionuclides and used forradionuclide therapy. Generally, it is believed that virtually any α,β-emitter, γ-emitter, or β, γ-emitter can be used in conjunction withthe invention. Preferred β, γ-emitters include ¹⁶⁶Ho, ¹⁸⁸Re, ¹⁸⁶Re,¹⁵³Sm, and ⁸⁹Sr. Preferred β-emitters include ⁹⁰Y and ²²⁵Ac. Preferredγ-emitters include ⁶⁷Ga, ⁶⁸Ga, ⁶⁴Cu, ⁶²Cu and ¹¹¹In. Preferredα-emitters include ²¹¹At and ²¹²Bi. It is also envisioned thatpara-magnetic substances, such as Gd, Mn and Fe can be chelated with ECfor use in conjunction with the present invention.

Complexes and means for preparing such complexes are convenientlyprovided in a kit form including a sealed vial containing apredetermined quantity of an EC-tissue specific ligand conjugate of theinvention to be labeled and a sufficient amount of reducing agent tolabel the conjugate with ^(99m)Tc. ^(99m)Tc labeled scintigraphicimaging agents according to the present invention can be prepared by theaddition of an appropriate amount of ^(99m)Tc or ^(99m)Tc complex into avial containing the EC-tissue specific ligand conjugate and reducingagent and reaction under conditions described in Example 1 hereinbelow.The kit may also contain conventional pharmaceutical adjunct materialssuch as, for example, pharmaceutically acceptable salts to adjust theosmotic pressure, buffers, preservatives, antioxidants, and the like.The components of the kit may be in liquid, frozen or dry form. In apreferred embodiment, kit components are provided in lyophilized form.

Radioactively labeled reagents or conjugates provided by the presentinvention are provided having a suitable amount of radioactivity. Informing ^(99m)Tc radioactive complexes, it is generally preferred toform radioactive complexes in solutions containing radioactivity atconcentrations of from about 0.01 millicurie (mCi) to about 300 mCi permL.

^(99m)Tc labeled scintigraphic imaging agents provided by the presentinvention can be used for visualizing sites in a mammalian body. Inaccordance with this invention, the ^(99m)Tc labeled scintigraphicimaging agents are administered in a single unit injectable dose. Any ofthe common carriers known to those with skill in the art, such assterile saline solution or plasma, can be utilized after radiolabelingfor preparing the injectable solution to diagnostically image variousorgans, tumors and the like in accordance with this invention.Generally, the unit dose to be administered has a radioactivity of about0.01 mCi to about 300 mCi, preferably 10 mCi to about 200 mCi. Thesolution to be injected at unit dosage is from about 0.01 mL to about 10mL. After intravenous administration, imaging of the organ or tumor invivo can take place, if desired, in hours or even longer, after theradiolabeled reagent is introduced into a patient. In most instances, asufficient amount of the administered dose will accumulate in the areato be imaged within about 0.1 of an hour to permit the taking ofscintiphotos. Any conventional method of scintigraphic imaging fordiagnostic or prognostic purposes can be utilized in accordance withthis invention.

The ^(99m)Tc-EC labeling strategy of the invention may also be used forprognostic purposes. It is envisioned that EC may be conjugated to knowndrugs of choice for cancer chemotherapy, such as those listed in Table2. These EC-drug conjugates may then be radio labeled with ^(99m)Tc andadministered to a patent having a tumor. The labeled EC-drug conjugateswill specifically bind to the tumor. Imaging may be performed todetermine the effectiveness of the cancer chemotherapy drug against thatparticular patient's particular tumor. In this way, physicians canquickly determine which mode of treatment to pursue, which chemotherapydrug will be most effective. This represents a dramatic improvement overcurrent methods which include choosing a drug and administering a roundof chemotherapy. This involves months of the patient's time and manythousands of dollars before the effectiveness of the drug can bedetermined.

The ^(99m)Tc labeled EC-tissue specific ligand conjugates and complexesprovided by the invention may be administered intravenously in anyconventional medium for intravenous injection such as an aqueous salinemedium, or in blood plasma medium. Such medium may also containconventional pharmaceutical adjunct materials such as, for example,pharmaceutically acceptable salts to adjust the osmostic pressure,buffers, preservatives, antioxidants and the like. Among the preferredmedia are normal saline and plasma.

Specific, preferred targeting strategies are discussed in more detailbelow.

Tumor Folate Receptor Targeting

The radiolabeled ligands, such as pentetreotide and vasoactiveintestinal peptide, bind to cell receptors, some of which areoverexpressed on tumor cells (Britton and Granowska, 1996; Krenning etal., 1995; Reubi et al., 1992; Goldsmith et al., 1995; Virgolini et al.,1994). Since these ligands are not immunogenic and are cleared quicklyfrom the plasma, receptor imaging would seem to be more promisingcompared to antibody imaging.

Folic acid as well as antifolates such as methotrexate enter into cellsvia high affinity folate receptors (glycosylphosphatidylinositol-linkedmembrane folate-binding protein) in addition to classical reduced-folatecarrier system (Westerhof et al., 1991; Orr et al., 1995; Hsuch andDolnick, 1993). Folate receptors (FRs) are overexposed on manyneoplastic cell types (e.g., lung, breast, ovarian, cervical,colorectal, nasopharyngeal, renal adenocarcinomas, malign melanoma andependymomas), but primarily expressed only several normal differentiatedtissues (e.g., choroid plexus, placenta, thyroid and kidney) (Orr etal., 1995; Weitman et al., 1992a; Campbell et al., 1991; Weitman et al.,1992b; Holm et al., 1994; Ross et al., 1994; Franklin et al., 1994;Weitman et al., 1994). FRs have been used to deliver folate-conjugatedprotein toxins, drug/antisense oligonucleotides and liposomes into tumorcells overexpressing the folate receptors (Ginobbi et al., 1997; Leamonand Low, 1991; Leamon and Low, 1992; Leamon et al., 1993; Lee and Low,1994). Furthermore, bispecific antibodies that contain anti-FRantibodies linked to anti-T cell receptor antibodies have been used totarget T cells to FR-positive tumor cells and are currently in clinicaltrials for ovarian carcinomas (Canevari et al., 1993; Bolhuis et al.,1992; Patrick et al., 1997; Coney et al., 1994; Kranz et al., 1995).Similarly, this property has been inspired to develop radiolabeledfolate-conjugates, such as ⁶⁷Ga-deferoxamine-folate and¹¹¹In-DTPA-folate for imaging of folate receptor positive tumors(Mathias et al., 1996; Wang et al., 1997; Wang et al., 1996; Mathias etal., 1997b). Results of limited in vitro and in vivo studies with theseagents suggest that folate receptors could be a potential target fortumor imaging. In this invention, the inventors developed a series ofnew folate receptor ligands. These ligands are ^(99m)Tc-EC-folate,^(99m)Tc-EC-methotrexate (^(99m)Tc-EC-MTX), ^(99m)Tc-EC-tomudex(^(99m)Tc-EC-TDX).

Tumor Hypoxia Targeting

Tumor cells are more sensitive to conventional radiation in the presenceof oxygen than in its absence; even a small percentage of hypoxic cellswithin a tumor could limit the response to radiation (Hall, 1988; Bushet al., 1978; Gray et al., 1953). Hypoxic radioresistance has beendemonstrated in many animal tumors but only in few tumor types in humans(Dische, 1991; Gatenby et al., 1988; Nordsmark et al., 1996). Theoccurrence of hypoxia in human tumors, in most cases, has been inferredfrom histology findings and from animal tumor studies. In vivodemonstration of hypoxia requires tissue measurements with oxygenelectrodes and the invasiveness of these techniques has limited theirclinical application.

Misonidazole (MISO) is a hypoxic cell sensitizer, and labeling MISO withdifferent radioisotopes (e.g., ¹⁸F, ¹²³I, ^(99m)Tc) may be useful fordifferentiating a hypoxic but metabolically active tumor from awell-oxygenated active tumor by PET or planar scintigraphy.[¹⁸F]Fluoromisonidazole (FMISO) has been used with PET to evaluatetumors hypoxia. Recent studies have shown that PET, with its ability tomonitor cell oxygen content through [¹⁸F]FMISO, has a high potential topredict tumor response to radiation (Koh et al., 1992; Valk et al.,1992; Martin et al., 1989; Rasey et al., 1989; Rasey et al., 1990; Yanget al., 1995). PET gives higher resolution without collimation, however,the cost of using PET isotopes in a clinical setting is prohibitive.Although labeling MISO with iodine was the choice, high uptake inthyroid tissue was observed. Therefore, it is desirable to developcompounds for planar scintigraphy that the isotope is less expensive andeasily available in most major medical facilities. In this invention,the inventors present the synthesis of ^(99m)Tc-EC-2-nitroimidazole and^(99m)Tc-EC-metronidazole and demonstrate their potential use as tumorhypoxia markers.

Peptide Imaging of Cancer

Peptides and amino acids have been successfully used in imaging ofvarious types of tumors (Wester et al., 1999; Coenen and Stocklin, 1988;Raderer et al., 1996; Lambert et al., 1990; Bakker et al., 1990; Stellaand Mathew, 1990; Butterfield et al., 1998; Piper et al., 1983;Mochizuki et al., Dickinson and Hiltner, 1981). Glutamic acid basedpeptide has been used as a drug carrier for cancer treatment (Stella andMathew, 1990; Butterfield et al., 1998; Piper et al., 1983; Mochizuki etal., 1985; Dickinson and Hiltner, 1981). It is known that glutamatemoiety of folate degraded and formed polyglutamate in vivo. Thepolyglutamate is then re-conjugated to folate to form folylpolyglutamate, which is involved in glucose metabolism. Labelingglutamic acid peptide may be useful in differentiating the malignancy ofthe tumors. In this invention, the inventors report the synthesis ofEC-glutamic acid pentapeptide and evaluate its potential use in imagingtumors.

Imaging Tumor Apoptotic Cells

Apoptosis occurs during the treatment of cancer with chemotherapy andradiation (Lennon et al., 1991; Abrams et al., 1990; Blakenberg et al.,1998; Blakenberg et al., 1999; Tait and Smith, 1991) Annexin V is knownto bind to phosphotidylserin, which is overexpressed by tumor apoptoticcells (Blakenberg et al., 1999; Tait and Smith, 1991). Assessment ofapoptosis by annexin V would be useful to evaluate the efficacy oftherapy such as disease progression or regression. In this invention,the inventors synthesize ^(99m)Tc-EC-annexin V (EC-ANNEX) and evaluateits potential use in imaging tumors.

Imaging Tumor Angiogenesis

Angiogenesis is in part responsible for tumor growth and the developmentof metastasis. Antimitotic compounds are antiangiogenic and are knownfor their potential use as anticancer drugs. These compounds inhibitcell division during the mitotic phase of the cell cycle. During thebiochemical process of cellular functions, such as cell division, cellmotility, secretion, ciliary and flagellar movement, intracellulartransport and the maintenance of cell shape, microtubules are involved.It is known that antimitotic compounds bind with high affinity tomicrotubule proteins (tubulin), disrupting microtubule assembly andcausing mitotic arrest of the proliferating cells. Thus, antimitoticcompounds are considered as microtubule inhibitors or as spindle poisons(Lu, 1995).

Many classes of antimitotic compounds control microtubuleassembly-disassembly by binding to tubulin (Lu, 1995; Goh et al., 1998;Wang et al., 1998; Rowinsky et al., 1990; Imbert, 1998). Compounds suchas colchicinoids interact with tubulin on the colchicine-binding sitesand inhibit microtubule assembly (Lu, 1995; Goh et al., 1998; Wang etal., 1998). Among colchicinoids, colchicine is an effectiveanti-inflammatory drug used to treat prophylaxis of acute gout.Colchicine also is used in chronic myelocytic leukemia. Althoughcolchicinoids are potent against certain types of tumor growth, theclinical therapeutic potential is limited due to inability to separatethe therapeutic and toxic effects (Lu, 1995). However, coichicine may beuseful as a biochemical tool to assess cellular functions. In thisinvention, the inventors developed ^(99m)Tc-EC-colchicine (EC-COL) forthe assessment of biochemical process on tubulin functions.

Imaging Tumor Apoptotic Cells

Apoptosis occurs during the treatment of cancer with chemotherapy andradiation. Annexin V is known to bind to phosphotidylserin, which isoverexpressed by tumor apoptotic cells. Assessment of apoptosis byannexin V would be useful to evaluate the efficacy of therapy such asdisease progression or regression. Thus, ^(99m)Tc-EC-annexin V(EC-ANNEX) was developed.

Imaging Tumor Hypoxia

The assessment of tumor hypoxia by an imaging modality prior toradiation therapy would provide rational means of selecting patients fortreatment with radiosensitizers or bioreductive drugs (e.g.,tirapazamine, mitomycin C). Such selection of patients would permit moreaccurate treatment patients with hypoxic tumors. In addition, tumorsuppressor gene (P53) is associated with multiple drug resistance. Tocorrelate the imaging findings with the overexpression of P53 byhistopathology before and after chemotherapy would be useful infollowing-up tumor treatment response. ^(99m)Tc-EC-2-nitroimidazole and^(99m)Tc-EC-metronidazole were developed.

Imaging Tumor Angiogenesis

Angiogenesis is in part responsible for tumor growth and the developmentof metastasis. Antimitotic compounds are antiangiogenic and are knownfor their potential use as anticancer drugs. These compounds inhibitcell division during the mitotic phase of the cell cycle. During thebiochemical process of cellular functions, such as cell division, cellmotility, secretion, ciliary and flagellar movement, intracellulartransport and the maintenance of cell shape, microtubules are involved.It is known that antimitotic compounds bind with high affinity tomicrotubule proteins (tubulin), disrupting microtubule assembly andcausing mitotic arrest of the proliferating cells. Thus, antimitoticcompounds are considered as microtubule inhibitors or as spindlepoisons. Colchicine, a potent antiangiogenic agent, is known to inhibitmicrotubule polymerization and cell arrest at metaphase. Colchicine(COL) may be useful as a biochemical tool to assess cellular functions.^(99m)Tc-EC-COL was then developed.

Imaging Hypoxia Due to Stroke

Although tumor cells are more or less hypoxic, it requires an oxygenprobe to measure the tensions. In order to mimic hypoxic conditions, theinventors imaged 11 patients who had experienced stroke using^(99m)Tc-EC-metronidazole (^(99m)Tc-EC-MN). Metronidazole is a tumorhypoxia marker. Tissue in the area of a stroke becomes hypoxic due tolack of oxygen. The SPECT images were conducted at 1 and 3 hours postinjection with ^(99m)Tc-EC-MN. All of these imaging studies positivelylocalized the lesions. CT does not show the lesions very well oraccurately. MRI and CT in some cases exaggerate the lesion size. Thefollowing are selected cases from three patients.

Case 1. A 59 year old male patient suffered a stroke in the left basalganglia. SPECT ^(99m)Tc-EC-MN identified the lesions at one hourpost-injection (FIG. 28), which corresponds to MRI T1 weighted image(FIG. 29).

Case 2. A 73 year old male patient suffered a stroke in the left mediumcerebral artery (MCA) territory. SPECT ^(99m)Tc-EC-MN was obtained atday 1 and day 12 (FIGS. 30 and 31) at one hour post-injection. Thelesions showed significant increased uptake at day 12. CT showedextensive cerebral hemorrhage in the lesions. No marked difference wasobserved between days 1 and 12 (FIGS. 32 and 33). The findings indicatethat the patient symptoms improved due to the tissue viability (fromanoxia to hypoxia). SPECT ^(99m)Tc-EC-MN provides functional informationwhich is better than CT images.

Case 3. A 72 year old male patient suffered a stroke in the right MCAand PCA area. SPECT ^(99m)Tc-EC-MN identified the lesions at one hourpost-injection (FIG. 34). CT exaggerates the lesion size. (FIG. 35).

Tumor Glycolysis Targeting

The radiolabeled ligands, such as polysaccharide (neomycin, kanamycin,tobramycin) and monosaccharide (glucosamine) bind to cell glucosetransporter, followed by phosphorylation which are overexpressed ontumor cells (Rogers et al., 1968; Fanciulli et al., 1994; Popovici etal., 1971; Jones et al., 1973; Hermann et al., 2000). Polysaccharide(neomycin, kanamycin, tobramycin) and monosaccharide (glucosamine)induced glucose level could be suppressed by insulin (Harada et al.,1995; Moller et al., 1991; Offield et al., 1996; Shankar et al., 1998;Yoshino et al., 1999; Villevalois-Cam et al., 2000) Since these ligandsare not immunogenic and are cleared quickly from the plasma, metabolicimaging would seem to be more promising compared to antibody imaging.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

EXAMPLE 1 Tumor Folate Receptor Targeting

Synthesis of EC

EC was prepared in a two-step synthesis according to the previouslydescribed methods (Ratner and Clarke, 1937; Blondeau et al., 1967; eachincorporated herein by reference). The precursor,L-thiazolidine-4-carboxylic acid, was synthesized (m.p. 195°, reported196-197°). EC was then prepared (m.p. 237°, reported 251-253°). Thestructure was confirmed by ¹H-NMR and fast-atom bombardment massspectroscopy (FAB-MS).

Synthesis of Aminoethylamido Analogue of Methotrexate (MTX-NH₂)

MIX (227 ma, 0.5 mmol) was dissolved in 1 ml of HCI solution (2N). ThepH value was <3. To this stirred solution, 2 ml of water and 4 ml ofN-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ, 6.609% inmethanol, 1 mmol) were added at room temperature. Ethylenediamine (EDA,0.6 ml, 10 mmol) was added slowly. The reaction mixture was stirredovernight and the solvent was evaporated in vacuo. The raw solidmaterial was washed with diethyl ether (10 ml), acetonitrile (10 ml) and95% ethyl alcohol (50 ml) to remove the unreacted EEDQ and EDA. Theproduct was then dried by lyophilization and used without furtherpurification. The product weighed 210 mg (84.7%) as a yellow powder.m.p. of product: 195-198° C. (dec, MIX); ¹H-NMR (D₂O) δ 2.98-3.04 (d,8H, —(CH₂)₂CONH(CH₀)₂NH₂), 4.16-4.71 (m, 6H, —CH₂. pteridinyl,aromatic-NCH₃, NH—CH—COOH glutamate), 6.63-6.64 (d, 2H, aromatic-CO),7.51-753 (d, 2H. aromatic-N), 8.36 (s, 1H, pteridinyl). FAB MS m/z calcdfor C₂₂H₂₈, N₁₀, O₄(M)⁺496.515, found 496.835.

Synthesis of Aminoethylamido Analogue of Folate (Folate-NH₂)

Folic acid dihydrate (1 g, 2.0 mmol) was added in 10 ml of water. The pHvalue was adjusted to 2 using HCI (2 N). To this stirred solution,N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ, 1 g in 10 mlmethanol, 4.0 mmol) and ethylenediamine (EDA, 1.3 ml, 18 mmol) wereadded slowly. The reaction mixture was stirred overnight at roomtemperature. The solvent was evaporated in vacuo. The product wasprecipitated in methanol (50 ml) and further washed with acetone (100ml) to remove the unreacted EEDQ and EDIT. The product was thenfreeze-dried and used without further purification. Ninhydrin (2% inmethanol) spray indicated the positivity of amino group. The productweighed 0.6 g (yield 60%) as a yellow powder. m.p. of product: 250°(dec). ¹H-NMR (D₂O) δ1.97-2.27 (m, 2H, —CH₂ glutamate of folate),3.05-3.40 (d, 6H, —CH₂CONH(CH₂)₂NH₂), 4.27-4.84 (m, 3H, —CH₂-pteridinyl,NH—CH—COOH glutamate), 6.68-6.70 (d, 2H, aromatic-CO), 7.60-7.62 (d, 2H,aromatic-N), 8.44 (s, 1H, pteridinyl). FAB MS m/z calcd for C₂₁H₂₅N₉,O₅(M)⁺ 483, found 483.21.

Synthesis of Ethylenedicysteine-Folate (EC-Folate)

To dissolve EC, NaOH (2N, 0.1 ml) was added to a stirred solution of EC(114 ma, 0.425 mmol) in water (1.5 ml). To this colorless solution,sulfo-NHS (92.3 mg, 0.425 mmol) and EDC (81.5 mg, 0.425 mmol) wereadded. Folate-NH₂ (205 mg, 0.425 mmol) was then added. The mixture wasstirred at room temperature for 24 hours. The mixture was dialyzed for48 hours using Spectra/POR molecular porous membrane with moleculecut-off at 500 (Spectrum Medical Industries Inc., Houston, Tex.). Afterdialysis, the product was freeze dried. The product weighed 116 mg(yield 35%). m.p. 195° (dec); ¹H-NMR (D₂O) δ1.98-2.28 (m, 2H, —CH2glutamate of folate), 2.60-2.95 (m, 4H and —CH₂—SH of EC). 3.24-3.34 (m,10H, —CH₂—CO, ethylenediamine of folate and ethylenediamine of EC),4.27-4.77 (m, 5H, —CH-pteridinyl, NH—CH—COOH glutamate of folate andNH—CH—COOH of EC), 6.60-6.62 (d, 2H, aromatic-CO), 7.58-7.59 (d, 2H.aromatic-N), 8.59 (s, 1H, pteridinyl). Anal. calcd for C29H37N₁₁S₂O₈Na₂(8H₂O), FAB MS m/z (M)⁺ 777.3 (free of water). C, 37.79; H. 5.75; N,16.72; S, 6.95. Found: m/z (M)⁺ 777.7 (20), 489.4 (100). C, 37.40; H,5.42; N. 15.43; S, 7.58.

Radiolabeling of EC-Folate and EC with ^(99m)Tc

Radiosynthesis of ^(99m)Tc-EC-folate was achieved by adding requiredamount of ^(99m)Tc-pertechnetate into home-made kit containing thelyophilized residue of EC-folate (3 mg), SnCl₂ (100 μg), Na_(2 HPO)₄(13.5 mg), ascorbic acid (0.5 mg) and NaEDTA (0.5 mg). Final pH ofpreparation was 7.4. ^(99m)Tc-EC was also obtained by using home-madekit containing the lyophilized residue of EC (3 mg), SnCl₂ (100 μg),Na₂, IPO₄ (13.5 mg), ascorbic acid (0.5 mg) and NaEDTA (0.5 mg) at pH10. Final pH of preparation was then adjusted to 7.4. Radiochemicalpurity was determined by TLC (ITLC SG, Gelman Sciences, Ann Arbor,Mich.) eluted with, respectively, acetone (system A) and ammoniumacetate (1M in water):methanol (4:1) (system B). From radio-TLC(Bioscan, Washington, D.C.) analysis, the radiochemical purity was >95%for both radiopharmaceuticals. Radio-TLC data are summarized in Table 2.Synthesis of ^(99m)Tc-EC-folate is shown in FIG. 1. TABLE 2 DRUGS OFCHOICE FOR CANCER CHEMOTHERAPY The tables that follow list drugs usedfor treatment of cancer in the USA and Canada and their major adverseeffects. The Drugs of Choice listing based on the opinions of MedicalLetter consultants. Some drugs are listed for indications for which theyhave not been approved by the US Food and Drug Administration.Anticancer drugs and their adverse effects follow. For purposes of thepresent invention, these lists are meant to be exemplary and notexhaustive. DRUGS OF CHOICE Cancer Drugs of Choice Some alternativesAdrenocortical** Mitotane Doxorubicin, streptozocin, Cisplatin etoposideBladder* Local: Instillation of BCG Instillation of mitomycin, Systemic:Methotrexate + vinblastine + doxorubicin + claplatin doxorubicin orthiotape (MVAC) Pecitaxel, substitution of Claplatin + Methotrexate +vinblastine carboplatin for claplatin in (CMV) combinations BrainAnaplastic astrocytoma* Procarbazine + lamuatine + vincristineCarmustine, Claplatin Anaplastic oligodendro- Procarbazine + lamustine +vincristine Carmustine, Claplatin Giloma* Gilabiastome** Carmustine orlamustine Procarbazine, claplatin Medulloblastoma Vincristine +carmustine ± mechiorethamine ± methotrexate Etoposide Mechiorethamine +vincristine + procarbazine + prednisone (MOPP) Vincristine + claplatin ±cyclophosphamide Primary central nervous Methotrexate (high doseIntravenous and/or system lymphoma Intrathecal) ± cytarabine(Intravenous and/or Intrathecal) Cyclophosphamide + Doxorubicin +vincristine + prednisone (CHOP) Breast Adjuvant¹: Cyclophosphamide +methotrexate + fluorouracil (CMF); Cyclophosphamide + Doxorubicin ±fluorouracil (AC or CAF); Tamoxifen Metastatic: Cyclophosphamide +methotrexate + fluorouracil Paclitaxel; thiotepa + Doxorubicin +vin-blastine; (CMF) or mitomycin + vinblastine; Cyclophosphamide +duxorubicin ± fluorouracil mitomycin + methotrexate + mitoxantrone; (ACor CAF) for receptor- fluorouracil by negative and/orhormone-refractory; continuous infusion; Bone Tamoxifen forreceptor-positive and/or marrow transplant³ hormone-sensitive² Cervix**Claplatin Chlorambucil, vincristine, Ifosfamide with means fluorouracil,Doxorubicin, Bleomycin + ifosfamide with means + claplatin methotrexate,altretamine Chorlocarcinoma Methotrexate ± leucovorin Methotrexate +dactinomycin + cyclophosphamide Dactinomycin (MAC) Etoposide +methotrexate + dactinomycin + cyclophosphamide + vincristine Colorectal*Adjuvant colon⁴: Fluorouracil + levam-isole; Hepatic metastases:fluorouracil + leucovorin Intrahepatic-arterial floxuridine Metastatic:fluorouracil + leucovorin Mitomycin Embryonal Vincristine + dectinomycin± cyclophasphamide Same + Doxorubicin rhabdomyosar-coma⁵ Vincristine +ifosfamide with means + etoposide Endometrial** Megastrol or anotherprogestin fluorouracil, tamoxifen, Doxorubicin + claplatin ±cyclophosphamide altretamine Esophageal* Claplatin + fluorouracilDoxorubicin, methotraxate, mitomycin Ewing's sarcoma⁵ Cyclophosphamide(or ifosfamide with CAV + etoposide means) + Doxorubicin + vincristine(CAV) ± dactinomycin Gastric** Fluorouracil ± leucavorin ClaplatinDoxorubicin, etoposide, methotrexate + leucovorin, mitomycin Head andneck Claplatin + fluorouracil Blomycin, carboplatin, paclitaxel squambuscell*⁶ Methotrexate Islet cell** Streptozocin + DoxorubicinStreptozocin + fluorouracil; chlorozotocin^(†); octreotide Kaposi'ssarcoma* Etoposide or interferon alfa or vinblastine Vincristine,Doxorubicin, (Aids-related) Doxorubicin + bleomycin + vincristine orbleomycin vinblastine (ABV) Leukemia Acute lymphocytic Induction:Vincristine + prednisone + asparaginase ± daunorubicin Induction: same ±high-dose leukemia CNS prophylaxis: Intrathecal methotrexate ± systemicmethotrexate ± cyterabine; (ALL)⁷ high-dose methotrexate withpegaspargase instead of leutovorin ± Intrathecal cytarabine ±Intrathecal asparaginese hydrocortisone Teniposide or etoposideHigh-dose cytarabine Maintenance: Methotrexate + mercaptopurineMaintenance: same + periodic Bone marrow transplant.³ ⁸ vincristine +prednisone Acute myeloid Induction: Cytsrabine + either daunorubicinCytarabine + mitoxentrone leukemia (AML)⁹ or idarubicin High-dosecyterabine Post Induction: High-dose cytarabine ± other drugs such asetoposide Bone marrow transplant³. Chronic lymphocytic Chlorambucil ±prednisone Cladribine, cyclophosphamide, leukemia (CLL) Fludarabinpentostatin, vincristine, Doxorubicin Chronic myeloid leukemia (CML)¹⁰Chronic phase Bone marrow transplant³ Busulfan Interferon alfaHydroxyures Accelerated¹¹ Bone marrow transplant³ Hydroxyures, busulfenBlast crisis¹¹ Lymphoid: Vincristine + prednisone + L- Tretinoln^(†)separaginess + intrathecal methotrexate (± maintenance Amsecrine,^(†)azacitidine with methotrexate + 8- Vincristine ± plicamycinmarcaptopurine) Hairy cell Leukemia Pentostatin or cladribine Interferonalfa, chlorambucil, fludarabin Liver** Doxorubicin Intrahepatic-arterialfloxuridine Fluorouracil or claplatin Lung, small cell (cat cell)Claplatin + etoposide (PE) Ifosfamide with means + carboplatin +etoposide Cyclophosphamide + doxorubicin + vincristine (ICE) (CAV) Dailyoral etoposide PE alternated with CAV Etoposide + ifosfamide withCyclophosphamide + etoposide + claplatin means + claplatin (VIP (CEP)Paclitaxel Duxorubicin + cyclophosphamide + etoposide (ACE) LungClaplatin + etoposide Claplatin + fluorouracil + leucovorin (non-smallcell)** Claplatin + Vinblastine ± mitomycin Carboplatin + paclitaxelClaplatin + vincrisine Lymphomas Hodgkin's¹² Doxorubicin + bleomycin +vinblastine + dacarbazine Mechlorethamine + vincristine + (ABVD)procarbazine + prednisone (MOPP) ABVD alternated with MOPPChlorambusil + vinblastine + procarbazine + prednisone ± carmustineMechlorethamine + vincristine + procarbazine Etoposide + vinblastine +doxorubicin (± prednisone) + doxorubicin + bleomycin + vinblastine Bonemarrow transplant³ (MOP[P]-ABV) Non-Hodgkin's Burkitt's lymphomaCyclophosphamide + vincristine + methotrexate Ifosfamide with meansCyclophosphamide + high-dose cytarabine ± methotrexateCyclophosphamide + doxorubicin + with leutovorin vincrletine +prednisone Intrathecal methotrexate or cytarabine (CHOP) Difuselarge-cell Cyclophosphamide + doxorubicin + Dexamethasone sometimeslymphoma vincristine + prednisone substituted for prednisone (CHOP)Other combination regimens, which may include methotrexate, etoposide,cytarabine, bleomycin, procarbazine, ifosfamide and mitoxantrone Bonemarrow transplant³ Follicular lymphoma Cyclophosphamide or chlorambusilSame ± vincristine and prednisone, ± etoposide Interferon alfa,cladribine, fludarabin Bone marrow transplant³ Cyclophosphamide +doxorubicin + vincristine + prednisone (CHOP) Melanoma** Interferon AlfaCarmustine, lomustine, cisplatin Dacarbazine Dacarbazine + clapletin +carmustine + tamoxifen Aldesleukin Mycosis fungoides* PUVA (psoralen +ultraviolet A) Isotretinoin, topical carmustine, Mechlorethamine(topical) pentosistin, fludarabin, Interferon alfa cladribine,photopheresis (extra- Electron beam radiotherapy corporealphotochemitherapy), Methotrexate chemotherapy as in non- Hodgkin'slymphoma Mysloma* Melphelan (or cyclophosphamide) + prednisonsInterferon alfa Melphalan ± carmustine + cyclophosphamide + prednisons +vincristine Bone marrow transplant³ Dexamethasone + doxorubicin +vincristine High-dose dexamethasons (VAD) Vincristine + carmustine +doxorubicin + prednisons (VBAP) Neuroblestoma* Doxorubicin +cyclophosphamide + claplatin + teniposide Carboplatin, etoposide oretoposide Bone marrow transplant³ doxorubicin + cyclophosphamideClaplatin + cyclophosphamide Osteogenic sarcoma⁵ Doxorubicin + claplatin± etopside ± ifosfamide Ifosfamide with means, etoposide, carboplatin,high- dose methotrexate with leucovorin Cyclophosphamide + etoposideOvary Claplatin (or carboplatin) + paclitaxel Ifosfamide with means,Claplatin (or carboplatin) + cyclophosphamide paclitaxel, tamoxifen,(CP) ± doxorubicin melphalan, altretamine (CAP) Pancreatic**Fluoroutacil ± leucovorin Gemoltabinet Prostate Leuprolide (orgoserelln) ± flutamide Estramustine ± vinblastine, aminoglutethimide +hydrocortleone, estramustine + etoposide, diethylstllbestrol, nilutamideRenal** Aldesleukin Vinblastine, floxuridine Inteferon alfaRetinoblestoma^(5*) Doxorubicin + cyclophosphamide ± claplatin ±etoposide ± vincristina Carboplatin, etoposide, Ifosfamide with meansSarcomas, soft Doxorubicin ± decarbazine ± cyclophosphamide ± IfosfamideMitornyeln + doxorubicin + claplatin tissue, adult* with meansVincristina, etoposide Testicular Claplatin + etoposide ± bleomycinVinblestine (or etoposide) + Ifosfamide (PEB) with means + claplatin(VIP) Bone marrow transplant³ Wilms' tumor⁵ Dectinomycln + vincriatine ±doxorubicin ± cyclophosphamide Ifosfamide with means, etoposide,carboplatin *Chemotherapy has only moderate activity. **Chemotherapy hasonly minor activity. ¹Tamoxifen with or without chemotherapy isgenerally recommended for postmenopausal estrogen-receptor-positive,mode-positive patients and chemotherapy with or without tamoxlfen forpremenopausal mode-positive patients. Adjuvant treatment withchemotherapy and/or tamoxifen is recommended for mode-negative patientswith larger tumors or other adverse prognostic indicators. ²Megastroland other hormonal agents may be effective in some patients withtamoxifen fails. ³After high-dose chemotherapy (Medical Letter, 34: 79,1982). ⁴For rectal cancer, postoperative adjuvant treatment withfluoroutacil plus radiation, preceded and followed by treatment withfluorouracil alone. ⁵Drugs have major activity only when combined withsurgical resection, radiotherapy or both. ⁶The vitamin A analoglactratinoln (Acgutana) can control pre-neoplastic lesions (leukoplakla)and decreases the rate of second primary tumors (SE Banner et al, J NatlCancer Inst, 88: 140 1994). ^(†)Available in the USA only forinvestigational use. ⁷High-risk patients (e.g., high counts, cytogeneticabnormalities, adults) may require additional drugs for induction,maintenance and “Intensificiation” (use of additional drugs afterachievement of remission). Additional drugs include cyclophosphamida,mitoxantrone and thloguanine. The results of one large controlled trialin the United Kingdom suggest that Intensificiation may improve survivalin all children with ALL (JM Chasselle et al, Lancet, 34B: 143, Jan. 21,1995). ⁸Patients with a poor prognosis initially or those who relapseafter remission. ⁹Some patients with acute promyelocytic leukemia havehad complete responses to tratinoin. Such treatment can cause a toxicsyndrome characterized primarily by fever and respiratory distress (RPWarrell, Jr et al, N Engl J Med. 328: 177, 1993). ¹⁰AllogeheicHLA-identical sibling bone marrow transplantation can cure 40% to 70% ofpatients with CML in chronic phase, 18% to 28% of patients withaccelerated phase CML, and <15% patients in blast crisis. Disease-freesurvival after bone marrow transplantations adversely influenced byage >50 years, duration of disease >3 years from diagnosis, and use ofone-antigen-mismatched or matched-unrelated donor marrow. Interferonalso may be curative in patients with chronic phase CML who achieve acomplete cytogenetic response (about 10%); it is the treatment of choicefor patents >80 years old with newly diagnosed chronic phase CML and forall patients who are not candidates for an allgensic bone marrowtransplant. Chemotherapy alone is palliative. ¹¹If a second chronicphase is achieved with any of these combinations, allogeneic bone marrowtransplant should be considered. Bone marrow transplant in secondchronic phase may be curative for 30% to 35% of patients with CML.¹²Limited-stage Hodgkin's disease (stages 1 and 2) is curable byradiotherapy. Disseminated disease (stages 3 b and 4) requirechemotherapy. Some intermediate stages and selected clinical situationsmay benefit from both. + Available in the USA only for investigationaluse. ANTICANCER DRUGS AND HORMONES Drug Acute Toxicity‡ Delayedtoxicity‡ Aldesleukin (Interleukin-2; Fever; fluid retention;hypertension; Neuropsychiatric disorders; Proleukin - Cetus respiratorydistress; rash; anemia; hypothyrldiam; nephrotic Oncology)thrombocytophenia; nausea and syndrome; possibly acute vomiting;diarrhea; capillary leak leukoencaphalopathy; syndrome; naphrotoxlolty;myocardialtoxicity; brachial plexopathy; bowel hepatotoxicity;erythernanodosum; perforation neutrophil chemotactic defects Altretamine(hexamethyl- Nausea and vomiting Bone marrow depression; melamine;Hexalen - U CNS depression; peripheral Bioscience) neuropathy; visualhallucinations; stexis; tremors, alopecia; rash AminogiutethimideDrowsiness; nausea; dizziness; rash Hypothryroidism (rare); bone(Cytadren - Ciba) marrow depression; fever; hypotension;mascullinization †Amsacrine (m-AMSA; Nausea and vomiting; diarrhea; painor Bone marrow depression; amaidine; AMSP P-D- phlebitis on infuelon;anaphylaxia hepactic injury; convulsions; Parke-Davis, Amsidyl-stomatitle; ventricular Warner-Lambert) fibrillation; alopecia;congestive heart failure; renal dysfunction Asparaginase (Elspar-merck;Nausea and vomiting; fever; chills; CNS depression or Kidrolase inCanada) headache; hypersensitivity, anaphylexia; hyperexcitability;acute abdominal pain; hyperglycemia leading hemorrhagic pancreatitis; tocoma coagulation defects; thromboals; renal damage; hepactic damageCervix** Claplatin Ifosfamide with means Chlorambucil, vincristine,Bleomycin patin fluoroutacil, doxorubicin, Ifosfamide with meansmethotrexete, altretamine Chorlocarcinoma Methotrexete ± leucovorinMethotrexete + dectinomycin + cyclophosphamide Dactinomyclin (MAC)Etoposide + methotrexate + dactinomycin + cyclophosphamide + vincrlatineColorectal* Adjuvant colon⁴: Fluoroutacil + lavamleole; Hepaticmetastases: fluoroutacil + leucovarin Intrahepactic-arterial Metastatic:Fluoroutacil + leucvarin floxuridine Mitomyclin Embryonal Vincriatine +dectinomycin ± cyclophosphamide Same + doxorubicin rhebdomyosarcoma⁶Vincristine + Ifosfamide with means + etoposide Endometrial** Megastrolor another progeetin Fluoroutacil, tamoxifen, Doxorubicin + claplatin ±cyclophosphamide altretamine Cancer Drugs of Choice Some alternativesEsophageal* Claplatin + Fluoroutacil Doxorubicin, methotrexete, Ewing'ssarcoma⁵ Cyclophosphamide (or ifosfamide with mitomycin means) +doxorubicin + vincrietine CAV + etoposide (CAV) ± dectinomycin Gastric**Fluoroutacil ± leucovoin Claplatin, doxorubicin, etoposide,methotrexete + leucovorin, mitomycin Head and neck squamous Claplatin +fluoroutacil Blaonycin, carboplatin, cell^(*5) Methotrexete paciltaxelIslet call** Streptozocin + doxorubicin Streptozocln + fluoroutacil;chlorozotocin; actreatide Kaposal's sercoma* Etoposide or Interferonalfa or Vincristine, doxorubicin, (AIDS-related) vinbleomycin stinebleomycln Doxorubicin + bleomycin + vincristine or vinbleomycin stine(ABV) Leukemias Induction: Vincristine + prednisone + asparaginase ±daunorubieln Industion: same ± high-dose Acute lymphocytic leukemia CNSprophylaxia; Intrathecal methotrexete ± cyterabine; (ALL)⁷ methotrexete± systemic high-dose pegaspargase instead of methotrexete withleucovorin ± Intrethecal aspareginese cytarabine ± IntrathecalTeniposide or etoposide hydrocortisone High-dose cytarabine Maintenance:methotrexete ± mercaptopurine Maintenance: same + periodic Bone marrowtransplant³ vincristine + prednisone Acute myeloid leukemia Induction:Cytarabine + either Cytarabine + mitoxantrone (AML)⁹ daunbrublein oridarubieln High-dose cytarabine Post Induction: High-dose cytarabine ±other drugs such as etoposide Bone marrow transplant³ Chroniclymophocytic Chlorambuell ± prednisone Claplatin, cyclophosphamide,leukemia (CLL) Fludarabin pentostatin, vinorlstine, doxorubicin†Available in the USA only for investigational use. ‡Dose-limitingeffects are in bold type. Cutaneous reactions (sometimes severe),hyperpigmentation, and ocular toxicity have been reported with virtuallyall nonhormonal anticancer drugs. For adverse interactions with otherdrugs, see the Medical Letter Handbook of Adverse Drug Interactions,1995. ¹Available in the USA only for investigational use. ²Megestrol andother hormonal agents may be effective in some pateients when tamoxifenfails. ³After high-dose chemotherapy (Medical Letter, 34: 78, 1992).⁴For rectal cancer, postoperative adjuvant treatment with fluoroutacilplus radiation, preceded and followed by treatment with fluoroutacilalone. ⁵Drugs have major activity only when combined with surgicalresection, radiotherapy or both. ⁶The vitamin A analog isotretinoin(Accutane) can control pre-neoplastic isions (leukoplaka) and decreasesthe rats of second primary tumors (SE Senner et al., J Natl Cancer Inst.88: 140, 1994). ⁷High-risk patients (e.g., high counts, cytogeneticabnormalities, adults) may require additionaldrugs for Induction,maintenance and “Intensification” (use of additional drugs afterachievement of remission). Additional drugs include cyclophosphamide,mitoxantrone and thioguamine. The results of one large controlled trialin the United Kingdom suggest that intensilibation may improve survivalin all children with ALL (jm Chassella et al., Lancet, 348: 143, Jan.21. 1998). ⁸Patients with a poor prognosis initially or those whorelapse after remission ⁹Some patients with acute promyclocytic leukemiahave had complete responses to tretinoin. Such treatment can cuase atoxic syndrome characterized primarily by fever and respiratory distress(RP Warrell, Jr et al. N Eng J. Med, 329: 177, 1993). ¹⁰Allogenaic HLAIdentical sibling bone marrow transplantation can cure 40% to 70% ofpatients with CML in chroni phase, 15% to 25% of patients withaccelerated phase CML, and <15% patients in blast crisis. Disease-freesurvival after bone marrow transplantation is adversely influenced byage >50 years, duration of disease >3 years from diagnosis, and use ofone antigen mismatched or matched-unrelated donor marrow. Inteferon alfamay be curative in patients with chronic phase CML who achieve acomplete cytogenetic resonse (about 10%); It is the treatment of choicesfor patients >50 years old with newly diagnosed chronic phase CML andfor all patients who are not candidates for an allogenic bone marrowtransplant. Chemotherapy alone is palliative.Radiolabeling of EC-MTX and EC-TDX with ^(99m)Tc

Use the same method described for the synthesis of EC-folate, EC-MTX andEC-TDX were prepared. The labeling procedure is the same as describedfor the preparation of ^(99m)Tc-EC-folate except EC-MTX and EC-TDX wereused. Synthesis of ^(99m)Tc-EC-MTX and ^(99m)Tc-EC-TDX is shown in FIG.2 and FIG. 3.

Stability Assay of ^(99m)Tc-EC-Folate, ^(99m)Tc-EC-MTX and^(99m)Tc-EC-TDX

Stability of ^(99m)Tc-EC-Folate, ^(99m)Tc-EC-MTX and ^(99m)Tc-EC-TDX wastested in serum samples. Briefly, 740 KBq of 1 mg ^(99m)Tc-EC-Folate,^(99m)Tc-EC-MIX and ^(99m)Tc-EC-TDX was incubated in dog serum (200 μl)at 37° C. for 4 hours. The serum samples was diluted with 50% methanolin water and radio-TLC repeated at 0.5, 2 and 4 hours as describedabove.

Tissue Distribution Studies

Female Fischer 344 rats (150±25 g) (Harlan Sprague-Dawley, Indianapolis,Ind.) were inoculated subcutaneously with 0.1 ml of mammary tumor cellsfrom the 13762 tumor cell line suspension (10⁶ cells/rat, a tumor cellline specific to Fischer rats) into the hind legs using 25-gaugeneedles. Studies performed 14 to 17 days after implantation when tumorsreached approximately 1 cm diameter. Animals were anesthetized withketamine (10-15 mg/rat, intraperitoneally) before each procedure.

In tissue distribution studies, each animal injected intravenously with370-550 KBq of ^(99m)Tc-EC-folate or ^(99m)Tc-EC (n=3/time point). Theinjected mass of each ligand was 10 μg per rat. At 20 min, 1, 2 and 4 hfollowing administration of the radiopharmaceuticals, the anesthetizedanimals were sacrificed and the tumor and selected tissues were excised,weighed and counted for radioactivity by a gamma counter (PackardInstruments, Downers Grove, Ill.). The biodistribution of tracer in eachsample was calculated as percentage of the injected dose per gram oftissue wet weight (% ID/g). Counts from a diluted sample of the originalinjectate were used for reference. Tumor/nontarget tissue count densityratios were calculated from the corresponding % ID/g values. Student-ttest was used to assess the significance of differences between twogroups.

In a separate study, blocking studies were performed to determinereceptor-mediated process. In blocking studies, for ^(99m)Tc-EC-folatewas co-administrated (i.v.) with 50 and 150 μmol/kg folic acid to tumorbearing rats (n=3/group). Animals were killed 1 h post-injection anddata was collected.

Scintigraphic Imaging and Autoradiography Studies

Scintigraphic images, using a gamma camera (Siemens Medical Systems,Inc., Hoffman Estates, Ill.) equipped with low-energy, parallel-holecollimator, were obtained 0.5, 2 and 4 hrs after i.v. injection of 18.5MBq of ^(99m)Tc-labeled radiotracer.

Whole-body autoradiogram were obtained by a quantitative image analyzer(Cyclone Storage Phosphor System, Packard, Meridian, Conn.). Followingi.v. injection of 37 MBq of ^(99m)Tc-EC-folate, animal killed at 1 h andbody was fixed in carboxymethyl cellulose (4%). The frozen body wasmounted onto a cryostat (LKB 2250 cryomicrotome) and cut into 100 μmcoronal sections. Each section was thawed and mounted on a slide. Theslide was then placed in contact with multipurpose phosphor storagescreen (MP, 7001480) and exposed for 15 h ^(99m)Tc-labeled). Thephosphor screen was excited by a red laser and resulting blue light thatis proportional with previously absorbed energy was recorded.

Results

Chemistry and Stability of ^(99m)Tc-EC-Folate

A simple, fast and high yield aminoethylamido and EC analogues offolate, MTX and TDX were developed. The structures of these analogueswere confirmed by NMR and mass spectroscopic analysis. Radiosynthesis ofEC-folate with ^(99m)Tc was achieved with high (>95%) radiochemicalpurity. ^(99m)Tc-EC-folate was found to be stable at 20 min. 1, 2 and 4hours in dog serum samples.

Biodistribution of ^(99m)Tc-EC-Folate

Biodistribution studies showed that tumor/blood count density ratios at20 min-4 h gradually increased for ^(99m)Tc-EC-folate, whereas thesevalues decreased for ^(99m)Tc-EC in the same time period (FIG. 4). %ID/g uptake values, tumor/blood and tumor/muscle ratios for^(99m)Tc-EC-folate and ^(99m)Tc-EC were given in Tables 3 and 4,respectively. TABLE 3 Biodistribution of ^(99m)Tc-EC-folate in BreastTumor-Bearing Rats % of injected ^(99m)Tc-EC-folate dose per organ ortissue 20 min 1 h 2 h 4 h Blood 0.370 ± 0.049 0.165 ± 0.028 0.086 ±0.005 0.058 ± 0.002 Lung 0.294 ± 0.017 0.164 ± 0.024 0.092 ± 0.002 0.063± 0.003 Liver 0.274 ± 0.027 0.185 ± 0.037 0.148 ± 0.042 0.105 ± 0.002Stomach 0.130 ± 0.002 0.557 ± 0.389 0.118 ± 0.093 0.073 ± 0.065 Kidney4.328 ± 0.896 4.052 ± 0.488 5.102 ± 0.276 4.673 ± 0.399 Thyroid 0.311 ±0.030 0.149 ± 0.033 0.095 ± 0.011 0.066 ± 0.011 Muscle 0.058 ± 0.0040.0257 ± 0.005  0.016 ± 0.007  0.008 ± 0.0005 Intestine 0.131 ± 0.0130.101 ± 0.071 0.031 ± 0.006 0.108 ± 0.072 Urine 12.637 ± 2.271  10.473 ±3.083  8.543 ± 2.763 2.447 ± 0.376 Tumor 0.298 ± 0.033 0.147 ± 0.0260.106 ± 0.029 0.071 ± 0.006 Tumor/Blood 0.812 ± 0.098 0.894 ± 0.0691.229 ± 0.325 1.227 ± 0.129 Tumor/Muscle 5.157 ± 0.690 5.739 ± 0.3476.876 ± 2.277 8.515 ± 0.307Values shown represent the mean ± standard deviation of data from 3animalsScintigraphic Imaging and Autoradiography Studies

Scintigraphic images obtained at different time points showedvisualization of tumor in ^(99m)Tc-EC-folate injected group. Contrary,there was no apparent tumor uptake in ^(99m)Tc-EC injected group (FIG.6). Both radiotracer showed evident kidney uptake in all images.Autoradiograms performed at 1 h after injection of ^(99m)Tc-EC-folateclearly demonstrated tumor activity.

EXAMPLE 2 Tumor Hypoxia Targeting Synthesis of 2-(2-methyl-5-nitro-¹Himidazolyl)ethylamine(amino analogue of metronidazole, MN-NH₂)

Amino analogue of metronidazole was synthesized according to thepreviously described methods (Hay et al., 1994) Briefly, metronidazolewas converted to a mesylated analogue (m.p. 149-150° C., reported153-154° C., TLC:ethyl acetate, Rf=0.45), yielded 75%. Mesylatedmetronidazole was then reacted with sodium azide to afford azidoanalogue (TLC:ethyl acetate, Rf=0.52), yielded 80%. The azido analoguewas reduced by triphenyl phosphine and yielded (60%) the desired aminoanalogue (m.p. 190-192° C., reported 194-195° C., TLC:ethyl acetate,Rf=0.15). Ninhydrin (2% in methanol) spray indicated the positivity ofamino group of MN—NH₂. The structure was confirmed by ¹H-NMR and massspectroscopy (FAB-MS) m/z 171(M⁺H, 100).

Synthesis of Ethylenedicysteine-Metronidazole (EC-MN)

Sodium hydroxide (2N, O.2 ml) was added to a stirred solution of EC (134ma, 0.50 mmol) in water (5 ml). To this colorless solution, sulfo-NHS(217 mg, 1.0 mmol) and 1˜)C (192 ma. 1.0 mmol) were added. MN-NH:dihydrochloride salt (340 mg, 2.0 mmol) was then added. The mature wasstirred at room temperature for 24 hours. The mixture was dialyzed for48 hrs using Spectra/POR molecular porous membrane with cut-off at 500(Spectrum Medical Industries Inc., Houston, Tex.). After dialysis, theproduct was frozen dried using lyophilizer (Labconco, Kansas City, Mo.).The product weighed 315 mg (yield 55%). ¹H-NMR (D₂0) δ 2.93 (s, 6H,nitroimidazole-CH ₃), 2.60-2.95 (m, 4H and —CH ₂—SH of EC), 3.30-3.66(m, 8H, ethylenediamine of EC and nitromidazole-CH₂—CH ₂—NH₂), 3.70-3.99(t, 2H, NH—CH—CO of EC), 5.05 (t, 4H, metronidazole-CH ₂—CH₂—NH₂) (s,2H, nitroimidazole C═CH). FAB MS m/z 572 (M⁺, 20). The synthetic schemeof EC-MN is shown in FIG. 7.

Synthesis of 3-(2-nitro-¹H-imidazolyl)propylamine (amino analogue ofnitroimidazole, NIM-NH₂)

To a stirred mixture containing 2-nitloimidazole (1 g, 8.34 mmol) andCs₂, CO₃ (2.9 g, 8.90 mmol) in dimethylformaide (DMF, 50 ml),1,3-ditosylpropane (3.84 g, 9.99 mmol) was added. The reaction washeated at 80° C. for 3 hours. The solvent was evaporated under vacuumand the residue was suspended in ethylacetate. The solid was filtered,the solvent was concentrated, loaded on a silica gel-packed column andeluted with hexane:ethylacetate (1:1). The product,3-tosylpropyl-(2-nitroimidazole), was isolated (1.67 g, 57.5%) with m.p.108-111° C. ¹H-NMR (CDCl₃) δ 2.23 (m, 2H), 2.48 (S. 3H), 4.06 (t, 2H,J=5.7 Hz), 4.52 (t, 2H, J=6.8 Hz), 7.09 (S. 1H), 7.24 (S. 1H), 7.40 (d,2H, J=8.2 Hz). 7.77 (d, 2H, J=8.2 Hz).

Tosylated 2-nitroimidazole (1.33 g, 4.08 mmol) was then reacted withsodium azide (Q29 g, 4.49 mmol) in DMF (10 ml) at 100° C. for 3 hours.After cooling, water (20 ml) was added and the product was extractedfrom ethylacetate (3×20 ml). The solvent was dried over MgSO₄ andevaporated to dryness to afford azido analogue (0.6 g, 75%, TLC:hexane:ethyl acetate; 1:1, Rf=0.42). ¹H-NMR (CDCl₃) δ 2.14 (m, 2H), 3.41(t, 2H, J=6.2 Hz), 4.54 (t, 2H, J=6.9 Hz), 7.17 (S. 2H).

The azido analogue (0.57 g, 2.90 mmol) was reduced by taphenyl phosphine(1.14 g, 4.35 mmol) in tetrahydrofuran (PHI;) at room temperature for 4hours. Concentrate HCI (12 ml) was added and heated for additional 5hours. The product was extracted from ethylacetate and water mixture.The ethylacetate was dried over MgSO₄ and evaporated to dryness toafford amine hydrochloride analogue (360 ma, 60%). Ninhydrin (2% inmethanol) spray indicated the positivity of amino group of NIM-NH.¹H-NMR (D₂O) δ 2.29 (m, 2H), 3.13 (t, 2H, J=7.8 Hz), 3.60 (br, 2H), 4.35(t, 2H, J=7.4 Hz), 7.50 (d, 1H, J=2.1 Hz), 7.63 (d, 1H, J=2.1 Hz).

Synthesis of Ethylenedicysteine-Nitroimidazole (EC-NIM)

Sodium hydroxide (2N, 0.6 ml) was added to a stirred solution of EC (134ma, 0.50 mmol) in water (2 ml). To this colorless solution, sulfo-NHS(260.6 mg, 1.2 mmol), EDC (230 ma, 1.2 mmol) and sodium hydroxide (2N, 1ml) were added. NIM-NH₂ hydrochloride salt (206.6 mg, 1.0 mmol) was thenadded. The mixture was stirred at room temperature for 24 hours. Themixture was dialyzed for 48 hrs using Spectra/POR molecular porousmembrane with cut-off at 500 (Spectrum Medical Industries Inc., Houston,Tex.). After dialysis, the product was frozen dried using lyophilizer(Labconco, Kansas City, Mo.). The product weighed 594.8 mg (yield 98%).The synthetic scheme of EC-NIM is shown in FIG. 8A. The structure isconfirmed by ¹H-NMR (D₂O) (FIG. 8B).

Radiolabeling of EC-MN and EC-NIM with ^(99m)Tc

Radiosynthesis of ^(99m)Tc-EC-MN and ^(99m)Tc-EC-NIM were achieved byadding required amount of pertechnetate into home-made kit containingthe lyophilized residue of EC-MN or EC-NIM (3 mg), SnCl₂, (100 μg),Na_(2 HPO) ₄ (13.5 mg), ascorbic acid (0.5 mg) and NaEDTA (0.5 mg).Final pH of preparation was 7.4. Radiochemical purity was determined byTLC (ITLAC SG, Gelman Sciences, Ann Arbor, Mich.) eluted with acetone(system A) and ammonium acetate (1M in water):methanol (4:1) (system B),respectively. From radio-TLC (Bioscan, Washington, D.C.) analysis, theradiochemical purity was >96% for both radiotracers.

Synthesis of [¹⁸F]FMISO and [¹³¹I]IMISO

[Should this be ¹⁸?][Fl]uoride was produced by the cyclotron usingproton irradiation of enriched ¹⁸O-water in a small-volume silvertarget. The tosyl MIS0 (Hay et al., 1994) (20 mg) was dissolved inacetonitrile (1.5 ml), added to the kryptofix-fluoride complex. Afterheating, hydrolysis and column purification, A yield of 25-40% (decaycorrected) of pure product was isolated with the end of bombardment(EOB) at 60 min. HPLC was performed on a C-18 ODS-20 T column, 4.6×25 mm(Waters Corp., Milford, Mass.), with water/acetonitrile, (80/20), usinga flow rate of 1 ml/min. The no-carrier-added product corresponded tothe retention time (6.12 min) of the unlabeled FMISO under similarconditions. The radiochemical purity was greater than 99%. Under the UVdetector (310 nm), there were no other impurities. The specific activityof [¹⁸F]FMISO determined was 1 Ci/μmol based upon UV and radioactivitydetection of a sample of known mass and radioactivity.

[¹³I]IMISO was prepared using the same precursor (Cherif et al., 1994),briefly, 5 mg of tosyl MISO was dissolved in acetonitrile (1 ml), andNa¹³¹I (1 mCi in 0.1 ml 1N NaOH) (Dupont New England Nuclear, Boston.MA) was added. After heating and purification, the product (60-70%yield) was obtained. Radio-TLC indicated the Rf values of 0.01 for thefinal product using chloroform methanol (7:3) as an eluant.

Stability Assay of ^(99m)Tc-EC-MN and ^(99m)Tc-EC-NIM

Stability of labeled ^(99m)Tc-EC-MN and ^(99m)Tc-EC-NIM were tested inserum samples. Briefly, 740 KBq of 1 mg ^(99m)Tc-EC-MN and^(99m)Tc-EC-NIM were incubated in dog serum (200 μl) at 37° C. for 4hours. The serum samples were diluted with 50% methanol in water andradio-TLC repeated at 0.5, 2 and 4 hours as described above.

Tissue Distribution Studies of ^(99m)Tc-EC-MN

Female Fischer 344 rats (150±25 g) (Harlan Sprague-Dawley, Indianapolis,Ind.) were inoculated subcutaneously with 0.1 ml of mammary tumor cellsfrom the 13762 tumor cell line suspension (10⁶ cells/rat, a tumor cellline specific to Fischer rats) into the hind legs using 25-gaugeneedles. Studies performed 14 to 17 days after implantation when tumorsreached approximately 1 cm diameter. Rats were anesthetized withketamine (10-15 mg/rat, intraperitoneally) before each procedure.

In tissue distribution studies, each animal was injected intravenouslywith 370-550 KBq of ^(99m)Tc-EC-MN or ^(99m)Tc-EC (n=3/time point). Theinjected mass of ^(99m)Tc-EC-MN was 10 μg per rat. At 0.5, 2 and 4 hrsfollowing administration of the radiotracers, the rats were sacrificedand the selected tissues were excised, weighed and counted forradioactivity. The biodistribution of tracer in each sample wascalculated as percentage of the injected dose per gram of tissue wetweight (% ID/g). Tumor/nontarget tissue count density radios werecalculated from the corresponding % ID/g values. The data was comparedto [¹⁸F]FMISO and [¹³¹I]IMISO using the same animal model. Studentt-test was used to assess the significance of differences betweengroups.

Scintigraphic Imaging and Autoradiography Studies

Scintigraphic images, using a gamma camera (Siemens Medical Systems,Inc., Hoffman Estates, Ill.) equipped with low-energy, parallel-holecollimator, were obtained 0.5, 2 and 4 hrs after i.v. injection of 18.5MBq of each radiotracer.

Whole-body autoradiogram was obtained by a quantitative image analyzer(Cyclone Storage Phosphor System, Packard, Meridian, Conn.). Followingi.v. injection of 37 MBq of ^(99m)Tc-EC-MN, the animals were killed at 1h and the body were fixed in carboxymethyl cellulose (4%) as previouslydescribed (Yang et al., 1995). The frozen body was mounted onto acryostat (LKB 2250 cryomicrotome) and cut into 100 μm coronal sections.Each section was thawed and mounted on a slide. The slide was thenplaced in contact with multipurpose phosphor storage screen (MP,7001480) and exposed for 15 hrs.

To ascertain whether ^(99m)Tc-EC-NIM could monitor tumor response tochemotherapy, a group of rats with tumor volume 1.5 cm and ovariantumor-bearing mice were treated with paclitaxel (40 mg/kg/rat, 80mg/kg/mouse, i.v.) at one single dose. The image was taken on day 4after paclitaxel treatment. Percent of injected dose per gram of tumorweight with or without treatment was determined.

Polarographic Oxygen Microelectrode pO₂ Measurements

To confirm tumor hypoxia, intratumoral pO₂ measurements were performedusing the Eppendorf computerized histographic system. Twenty totwenty-five pO₂ measurements along each of two to three linear trackswere performed at 0.4 mm intervals on each tumor (40-75 measurementstotal). Tumor pO measurements were made on three tumor-bearing rats.Using an on-line computer system, the pot measurements of each trackwere expressed as absolute values relative to the location of themeasuring point along the track, and as the relative frequencies withina pO₂ histogram between 0 and 100 mmHg with a class width of 2.5 mm.

Results

Radiosynthesis and Stability of ^(99m)Tc-EC-MN and ^(99m)Tc-EC-NIM

Radiosynthesis of EC-MN and EC-NIM with ^(99m)Tc were achieved with high(>95%) radiochemical purity Radiochemical yield was 100%. ^(99m)Tc-EC-MNand ^(99m)Tc-EC-NIM (FIG. 13) were found to be stable at 0.5, 2 and 4hrs in dog serum samples. There was no degradation products observed.Radiofluorination and radioiodination of MISO were achieved easily usingthe same precursor. In both labeled MISO analogues, the radiochemicalpurity was greater than 99%.

In Vivo Tissue Distribution Studies

The tissue distribution of ^(99m)Tc-EC-MN and ^(99m)Tc-EC in thetumor-bearing rats is shown in Tables 4 and 5. Due to high affinity forionic ^(99m)Tc, there was no significant and consistent thyroid uptake,suggesting the in vivo stability of ^(99m)Tc-EC-MN (Table 5). TABLE 4Biodistribution of ^(99m)Tc-EC in Breast Tumor-Bearing Rats % ofinjected ^(99m)Tc-EC dose per organ or tissue 20 min 1 h 2 h 4 h Blood0.435 ± 0.029 0.273 ± 0.039 0.211 ± 0.001 0.149 ± 0.008 Lung 0.272 ±0.019 0.187 ± 0.029 0.144 ± 0.002 0.120 ± 0.012 Liver 0.508 ± 0.0620.367 ± 0.006 0.286 ± 0.073 0.234 ± 0.016 Stomach 0.136 ± 0.060 0.127 ±0.106 0.037 ± 0.027 0.043 ± 0.014 Kidney 7.914 ± 0.896 8.991 ± 0.2689.116 ± 0.053 7.834 ± 1.018 Thyroid 0.219 ± 0.036 0.229 ± 0.118 0.106 ±0.003 0.083 ± 0.005 Muscle 0.060 ± 0.006 0.043 ± 0.002 0.028 ± 0.0090.019 ± 0.001 Intestine 0.173 ± 0.029 0.787 ± 0.106 0.401 ± 0.093 0.103± 0.009 Urine 9.124 ± 0.808 11.045 ± 6.158  13.192 ± 4.505  8.693 ±2.981 Tumor 0.342 ± 0.163 0.149 ± 0.020 0.115 ± 0.002 0.096 ± 0.005Tumor/Blood 0.776 ± 0.322 0.544 ± 0.004 0.546 ± 0.010 0.649 ± 0.005Tumor/Muscle 5.841 ± 3.253 3.414 ± 0.325 4.425 ± 1.397 5.093 ± 0.223Values shown represent the mean ± standard deviation of data from 3animals

In blocking studies, tumor/muscle and tumor/blood count density ratioswere significantly decreased (p<0.01) with folic acid co-administrations(FIG. 5). TABLE 5 Biodistribution of ^(99m)Tc-EC-metronidazole conjugatein breast tumor bearing rats¹ 30 Min. 2 Hour 4 Hour Blood 1.46 ± 0.731.19 ± 0.34 0.76 ± 0.14 Lung 0.79 ± 0.39 0.73 ± 0.02 0.52 ± 0.07 Liver0.83 ± 0.36 0.91 ± 0.11 0.87 ± 0.09 Spleen 0.37 ± 0.17 0.41 ± 0.04 0.37± 0.07 Kidney 4.30 ± 1.07 5.84 ± 0.43 6.39 ± 0.48 Muscle 0.08 ± 0.030.09 ± 0.01 0.07 ± 0.01 Intestine 0.27 ± 0.12 0.39 ± 0.24 0.22 ± 0.05Thyroid 0.051 ± 0.16  0.51 ± 0.09 0.41 ± 0.02 Tumor 0.034 ± 0.13  0.49 ±0.02 0.50 ± 0.09¹Each rat received 99m Tc-EC-metronidazole (10 μCi, iv). Each value ispercent of injected dose per gram weight (n = 3)/time interval. Eachdata represents mean of three measurements with standard deviation.

Biodistribudon studies showed that tumor/blood and tumor/muscle countdensity ratios at 0.54 hr gradually increased for ^(99m)Tc-EC-MN,[¹⁸F]FMISO and [¹³¹I]IMISO, whereas these values did not alter for^(99m)Tc-EC in the same time period (FIG. 9 and FIG. 10). [¹⁸F]FMISOshowed the highest tumor-to-blood uptake ratio than those with[¹³¹I]IMISO and ^(99m)Tc-EC-MN at 30 min, 2 and 4 hrs post-injection.Tumor/blood and tumor/muscle ratios for ^(99m)Tc-EC-MN and [¹³¹I]IMISOat 2 and 4 hrs postinjection were not significantly different (p<0.05).

Scintigraphic Imaging and Autoradiographic Studies

Scintigraphic images obtained at different time points showedvisualization of tumor in ^(99m)Tc-EC-MN and ^(99m)Tc-EC-NIM groups.Contrary, there was no apparent tumor uptake in ^(99m)Tc-EC injectedgroup (FIG. 11). Autoradiograms performed at 1 hr after injection of^(99m)Tc-EC-MN clearly demonstrated tumor activity (FIG. 12). Compare to^(99m)Tc-EC-NM, ^(99m)Tc-EC-NIM appeared to provide better scintigraphicimages due to higher tumor-to-background ratios. In breast tumor-bearingrats, tumor uptake was markedly higher in ^(99m)Tc-EC-NIM group comparedto ^(99m)Tc-EC (FIG. 14A). Data obtained from percent of injected doseof ^(99m)Tc-EC-NIM per gram of tumor weight indicated that a 25%decreased uptake in the rats treated with paclitaxel when compared tocontrol group (FIG. 14B).

In ovarian tumor-bearing mice, there was a decreased tumor uptake inmice treated with paclitaxel (FIG. 15A and FIG. 15B). Similar resultswere observed in sarcoma-bearing (FIG. 15C and FIG. 15D). Thus,^(99m)Tc-EC-NIM could be used to assess tumor response to paclitaxeltreatment.

Polarographic Oxygen Microelectrode pO₂ Measurements

Intratumoral PO₂ measurements of tumors indicated the tumor oxygentension ranged 4.6±1.4 mmHg as compared to normal muscle of 35±10 mmHg.The data indicate that the tumors are hypoxic.

EXAMPLE 3 Peptide Imaging of Cancer

Synthesis of Ethylenedicysteine-Pentaglutamate (EC-GAP)

Sodium hydroxide (1N, 1 ml) was added to a stirred solution of EC (200mg, 0.75 mmol) in water (10 ml). To this colorless solution, sulfo-NHS(162 mg, 0.75 mmol) and EDC (143 mg, 0.75 mmol) were added.Pentaglutamate sodium salt (M.W. 750-1500, Sigma Chemical Company) (500mg, 0.67 mmol) was then added. The mixture was stirred at roomtemperature for 24 hours. The mixture was dialyzed for 48 hrs usingSpectra/POR molecular porous membrane with cut-off at 500 (SpectrumMedical Industries Inc., Houston, Tex.). After dialysis, the product wasfrozen dried using lyophilizer (Labconco, Kansas City, Mo.). The productin the salt form weighed 0.95 g. The synthetic scheme of EC-GAP is shownin FIG. 16.

Stability Assay of ^(99m)Tc-EC-GAP

Radiolabeling of EC-GAP with ^(99m)Tc was achieved using the sameprocedure described previously. The radiochemical purity was 100%.Stability of labeled ^(99m)Tc-EC-GAP was tested in serum samples.Briefly, 740 KBq of 1 mg ^(99m)Tc-EC-GAP was incubated in dog serum (200μl) at 37° C. for 4 hours. The serum samples were diluted with 50%methanol in water and radio-TLC repeated at 0.5, 2 and 4 hours asdescribed above.

Scintigraphic Imaging Studies

Scintigraphic images, using a gamma camera equipped with low-energy,parallel-hole collimator, were obtained 0.5, 2 and 4 hrs after i.v.injection of 18.5 MBq of each radiotracer.

Results

Stability Assay of ^(99m)Tc-EC-GAP

^(99m)Tc-EC-GAP found to be stable at 0.5, 2 and 4 hrs in dog serumsamples. There was no degradation products observed.

Scintigraphic Imaging Studies

Scintigraphic images obtained at different time points showedvisualization of tumor in ^(99m)Tc-EC-GAP group. The optimum uptake isat 30 min to 1 hour post-administration (FIG. 17).

EXAMPLE 4 Imaging Tumor Apoptotic Cells

Synthesis of Ethylenedicysteine-Annexin V (EC-ANNEX)

Sodium bicarbonate (1N, 1 ml) was added to a stirred solution of EC (5mg, 0.019 mmol). To this colorless solution, sulfo-NHS (4 mg, 0.019mmol) and EDC (4 mg, 0.019 mmol) were added. Annexin V (M.W. 33 kD,human, Sigma Chemical Company) (0.3 mg) was then added. The mixture wasstirred at room temperature for 24 hours. The mixture was dialyzed for48 hrs using Spectra/POR molecular porous membrane with cut-off at10,000 (Spectrum Medical Industries Inc., Houston, Tex.). Afterdialysis, the product was frozen dried using lyophilizer (Labconco,Kansas City, Mo.). The product in the salt form weighed 12 mg.

Stability Assay of ^(99m)Tc-EC-ANNEX

Radiolabeling of EC-ANNEX with ^(99m)Tc was achieved using the sameprocedure described in EC-GAP. The radiochemical purity was 100%.Stability of labeled ^(99m)Tc-EC-ANNEX was tested in serum samples.Briefly, 740 KBq of 1 mg ^(99m)Tc-EC-ANNEX was incubated in dog serum(200 μl) at 37° C. for 4 hours. The serum samples were diluted with 50%methanol in water and radio-TLC repeated at 0.5, 2 and 4 hours asdescribed above.

Scintigraphic Imaging Studies

Scintigraphic images, using a gamma camera equipped with low-energy,parallel-hole collimator, were obtained 0.5, 2 and 4 hrs after i.v.injection of 18.5 MBq of the radiotracer. The animal models used werebreast, ovarian and sarcoma. Both breast and ovarian-tumor bearing ratsare known to overexpress high apoptotic cells. The imaging studies wereconducted on day 14 after tumor cell inoculation. To ascertain the tumortreatment response, the pre-imaged mice were administered paclitaxel (80mg/Kg, iv, day 14) and the images were taken on day 18.

Results

Stability Assay of ^(99m)Tc-EC-ANNEX

^(99m)Tc-EC-ANNEX found to be stable at 0.5, 2 and 4 hrs in dog serumsamples. There was no degradation products observed.

Scintigraphic Imaging Studies

Scintigraphic images obtained at different time points showedvisualization of tumor in ^(99m)Tc-EC-ANNEX group (FIGS. 18-20). Theimages indicated that highly apoptotic cells have more uptake of^(99m)Tc-EC-ANNEX. There was no marked difference of tumor uptakebetween pre- and post-[aclitaxel treatment in the high apoptosis(ovarian tumor-bearing) group (FIG. 19A and FIG. 19B) and in the lowapoptosis (sarcoma tumor-bearing) group (FIG. 20A and FIG. 20B).

EXAMPLE 5 Imaging Tumor Angiogenesis

Synthesis of (Amino Analogue of Colchcine, COL-NH₂)

Demethylated amino and hydroxy analogue of colchcine was synthesizedaccording to the previously described methods (Orr et al., 1995).Briefly, colchicine (4 g) was dissolved in 100 ml of water containing25% sulfuric acid. The reaction mixture was heated for 5 hours at 100°C. The mixture was neutralized with sodium carbonate. The product wasfiltered and dried over freeze dryer, yielded 2.4 g (70%) of the desiredamino analogue (m.p. 153-155° C., reported 155-157° C.). Ninhydrin (2%in methanol) spray indicated the positivity of amino group of COL-NH₂.The structure was confirmed by ¹H-NMR and mass spectroscopy (FAB-MS).¹H-NMR (CDCl₃)δ 8.09 (S, 1H), 7.51 (d, 1H, J=12 Hz), 7.30 (d, 1H, J=12Hz), 6.56 (S, 1H), 3.91 (S, 6H), 3.85 (m, 1H), 3.67 (S, 3H), 2.25-2.52(m, 4H). m/z 308.2(M⁺, 20), 307.2 (100).

Synthesis of Ethylenedicysteine-Colchcine (EC-COL)

Sodium hydroxide (2N, 0.2 ml) was added to a stirred solution of EC (134mg, 0.50 mmol) in water (5 ml). To this colotiess solution, sulfo-NHS(217 mg, 1.0 mmol) and EDC (192 mg, 1.0 mmol) were added. COL-NH₂ (340mg, 2.0 mmol) was then added. The mixture was stirred at roomtemperature for 24 hours. The mixture was dialyzed for 48 hrs usingSpectra/POR molecular porous membrane with cut-off at 500 (SpectrumMedical Industries Inc., Houston, Tex.). After dialysis, the product wasfrozen dried using lyophilizer (Labconco, Kansas City, Mo.). The productweighed 315 mg (yield 55%). ¹H-NMR (D₂O) δ 7.39 (S, 1H), 7.20 (d, 1H,J=12 Hz), 7.03 (d, 1H, J=12 Hz), 6.78 (S, 1H), 4.25-4.40 (m, 1H), 3.87(S, 3H, —OCH₃), 3.84 (S, 3H, —OCH₃), 3.53 (S, 3H, —OCH₃), 3.42-3.52 (m,2H), 3.05-3.26 (m, 4H), 2.63-2.82 (m, 4H), 2.19-2.25 (m, 4H). FAB MS m/z580 (sodium salt, 20). The synthetic scheme of EC-COL is shown in FIG.21.

Radiolabeling of EC-COL and EC with ^(99m)Tc

Radiosynthesis of ^(99m)Tc-EC-COL was achieved by adding required amountof ^(99m)Tc-pertechnetate into home-made kit containing the lyophilizedresidue of EC-COL (5 mg), SnCl₂ (100 μg), Na_(2 HPO) ₄ (13.5 mg),ascorbic acid (0.5 mg) and NaEDTA (0.5 mg). Final pH of preparation was7.4. ^(99m)Tc-EC was also obtained by using home-made kit containing thelyophilized residue of EC (5 mg), SnCl₂ (100 μg), Na_(2 HPO) ₄ (13.5mg), ascorbic acid (0.5 mg) and NaEDTA (0.5 mg) at pH 10. Final pH ofpreparation was then adjusted to 7.4. Radiochemical purity wasdetermined by TLC (ITLC SG, Gelman Sciences, Ann Arbor, Mich.) elutedwith ammonium acetate (1M in water):methanol (4:1). Radio-thin layerchromatography (TLC, Bioscan, Washington, D.C.) was used to analyze theradiochemical purity for both radiotracers.

Stability Assay of ^(99m)Tc-EC-COL

Stability of labeled ^(99m)Tc-EC-COL was tested in serum samples.Briefly, 740 KBq of 5 mg ^(99m)Tc-EC-COL was incubated in the rabbinateserum (500 μl) at 37° C. for 4 hours. The serum samples was diluted with50% methanol in water and radio-TLC repeated at 0.5, 2 and 4 hours asdescribed above.

Tissue Distribution Studies

Female Fischer 344 rats (150±25 g) (Harlan Sprague-Dawley, Indianapolis,Ind.) were inoculated subcutaneously with 0.1 ml of mammary tumor cellsfrom the 13762 tumor cell line suspension (10 cells/rat, a tumor cellline specific to Fischer rats) into the hind legs using 25-gaugeneedles. Studies performed 14 to 17 days after implantation when tumorsreached approximately 1 cm diameter. Rats were anesthetized withketamine (10-15 mg/rat, intraperitoneally) before each procedure.

In tissue distribution studies, each animal was injected intravenouslywith 370-550 KBq of ^(99m)Tc-EC-COL or ^(99m)Tc-EC (n=3/time point). Theinjected mass of ^(99m)Tc-EC-COL was 10 μg per rat. At 0.5, 2 and 4 hrsfollowing administration of the radiotracers, the rats were sacrificedand the selected tissues were excised, weighed and counted forradioactivity. The biodistribution of tracer in each sample wascalculated as percentage of the injected dose per gram of tissue wetweight (% ID/g). Tumor/nontarget tissue count density ratios werecalculated from the corresponding % ID/g values. Student t-test was usedto assess the significance of differences between groups.

Scintigraphic Imaging Studies

Scintigraphic images, using a gamma camera (Siemens Medical Systems,Inc., Hoffman Estates, Ill.) equipped with low-energy, parallel-holecollimator, were obtained 0.5, 2 and 4 hrs after i.v. injection of 300μCi of ^(99m)Tc-EC-COL and ^(99m)Tc-EC. Computer outlined region ofinterest (ROI) was used to quantitate (counts per pixel) the tumoruptake versus normal muscle uptake.

Results

Radiosynthesis and Stability of ^(99m)Tc-EC-COL

Radiosynthesis of EC-COL with ^(99m)Tc was achieved with high (>95%)radiochemical purity (FIG. 21). ^(99m)Tc-EC-COL was found to be stableat 0.5, 2 and 4 hrs in rabbit serum samples. There was no degradationproducts observed (FIG. 22).

In Vivo Biodistribution

In vivo biodistribution of ^(99m)Tc-EC-COL and ^(99m)Tc-EC inbreast-tumor-bearing rats are shown in Tables 4 and 6. Tumor uptakevalue (% ID/g) of ^(99m)Tc-EC-COL at 0.5, 2 and 4 hours was 0.436±0.089,0.395±0.154 and 0.221±0.006 (Table 6), whereas those for ^(99m)Tc-ECwere 0.342±0.163, 0.115±0.002 and 0.097±0.005, respectively (Table 4).Increased tumor-to-blood (0.52±0.12 to 0.72±0.07) and tumor-to-muscle(3.47±0.40 to 7.97±0.93) ratios as a function of time were observed in^(99m)Tc-EC-COL group (FIG. 23). Conversely, tumor-to-blood andtumor-to-muscle values showed time-dependent decrease with ^(99m)Tc-ECwhen compared to ^(99m)Tc-EC-COL group in the same time period (FIG.24). TABLE 6 Biodistribution of ^(99m)Tc-EC-Colchicine in Breast TumorBearing Rats 30 Min. 2 Hour 4 Hour Blood 0.837 ± 0.072 0.606 ± 0.2660.307 ± 0.022 Lung 0.636 ± 0.056 0.407 ± 0.151 0.194 ± 0.009 Liver 1.159± 0.095 1.051 ± 0.213 0.808 ± 0.084 Spleen 0.524 ± 0.086 0.559 ± 0.1430.358 ± 0.032 Kidney 9.705 ± 0.608 14.065 ± 4.007  11.097 ± 0.108 Muscle 0.129 ± 0.040 0.071 ± 0.032 0.028 ± 0.004 Stomach 0.484 ± 0.3860.342 ± 0.150 0.171 ± 0.123 Uterus 0.502 ± 0.326 0.343 ± 0.370 0.133 ±0.014 Thyroid 3.907 ± 0.997 2.297 ± 0.711 1.709 ± 0.776 Tumor 0.436 ±0.089 0.395 ± 0.154 0.221 ± 0.006*Each rat received ^(99m)Tc-EC-Colchicine (10 μCi, iv.). Each value isthe percent of injected dose per gram tissue weight (n = 3)/timeinterval. Each data represents mean of three measurements with standarddeviation.

TABLE 7 Rf Values Determined by Radio-TLC (ITLC-SG) Studies System A*System B† ^(99m)Tc-EC-folate 0 1(>95%) ^(99m)Tc-EC- 0 1(>95%) Free^(99m)TC 1 1 Reduced ^(99m)TC 0 0*Acetone^(†)Ammonium Acetate (1M in water):Methanol (4:1)

Gamma Scintigraphic Imaging of ^(99m)Tc-EC-COL in Breast Tumor-BearingRats

In vivo imaging studies in three breast-tumor-bearing rats at 1 hourpost-administration indicated that the tumor could be visualized wellwith ^(99m)Tc-EC-COL group (FIG. 25), whereas, less tumor uptake in the^(99m)Tc-EC group was observed (FIG. 26). Computer outlined region ofinterest (ROI) showed that tumor/background ratios in ^(99m)Tc-EC-COLgroup were significantly higher than ^(99m)Tc-EC group) (FIG. 27).

Tumor Glycolysis Targeting

EXAMPLE 6 Development of ^(99m)Tc-EC-Neomycin

Synthesis of EC

EC was prepared in a two-step synthesis according to the previouslydescribed methods (Ratner and Clarke, 1937; Blondeau et al., 1967). Theprecursor, L-thiazolidine-4-carboxylic acid, was synthesized (m.p. 1950,reported 196-197°). EC was then prepared (m.p. 237°, reported 251-253°).The structure was confirmed by ¹H-NMR and fast-atom bombardment massspectroscopy (FAB-MS).

Synthesis of Ethylenedicysteine-Neomycin (EC-Neomycin)

Sodium hydroxide (2N, 0.2 ml) was added to a stirred solution of EC (134mg, 0.50 mmol) in water (5 ml). To this colorless solution, sulfo-NHS(217 mg, 1.0 mmol) and EDC (192 mg, 1.0 mmol) were added. Neomycintrisulfate salt (909 mg, 1.0 mmol) was then added. The mixture wasstirred at room temperature for 24 hours. The mixture was dialyzed for48 hours using Spectra/POR molecular porous membrane with cut-off at 500(Spectrum Medical Industries Inc., Houston, Tex.). After dialysis, theproduct was frozen dried using lyophilizer (Labconco, Kansas City, Mo.).The product weighed 720 mg (yield 83%). The synthetic scheme ofEC-neomycin is shown in FIG. 36. The structure is confirmed by ¹H-NMR(FIGS. 38A-B), mass spectrometry (FIGS. 39A-B) and elemental analysis(Galbraith Laboratories, Inc. Knoxyille, Tenn.). Elemental analysisC₃₉H₇₅N₁₀S₄O₁₉.15H₂O(C, H, N, S), Calc. C, 33.77; H, 7.58; N: 10.11, S:9.23; found C, 32.44; H, 5.90; N, 10.47, S: 10.58. UV wavelength ofEC-neomycin was shifted to 270.5 nm when compared to EC and neomycin(FIGS. 40A-C)

Radiolabeling of EC-MN and EC-Neomycin with ^(99m)Tc

Radiosynthesis of ^(99m)Tc-EC and ^(99m)Tc-EC-neomycin were achieved byadding required amount of ^(99m)Tc-pertechnetate into home-made kitcontaining the lyophilized residue of EC or EC-neomycin (10 mg), SnCl₂(100 μg), Na_(2 HPO) ₄ (13.5 mg) and ascorbic acid (0.5 mg). NaEDTA (0.5mg) in 0.1 ml of water was then added. Final pH of preparation was 7.4.Radiochemical purity was determined by TLC (ITLC SG, Gelman Sciences,Ann Arbor, Mich.) eluted with ammonium acetate (1M in water):methanol(4:1). From radio-TLC (Bioscan, Washington, D.C.) analysis (FIG. 41) andHPLC analysis (FIGS. 42-45), the radiochemical purity was >95% for bothradiotracers.

Stability Assay of ^(99m)Tc-EC and ^(99m)Tc-EC-Neomycin

Stability of labeled ^(99m)Tc-EC and ^(99m)Tc-EC-neomycin were tested indog serum samples. Briefly, 740 KBq of 1 mg ^(99m)Tc-EC and^(99m)Tc-EC-neomycin were incubated in dog serum (200 μl) at 37° C. for4 hours. The serum samples were diluted with 50% methanol in water andradio-TLC repeated at 0.5, 2 and 4 hours as described above.

Tissue Distribution Studies of ^(99m)Tc-EC-Neomycin

Female Fischer 344 rats (150±25 g) (Harlan Sprague-Dawley, Indianapolis,Ind.) were innoculated subcutaneously with 0.1 ml of mammary tumor cellsfrom the 13762 tumor cell line suspension (10⁶ cells/rat, a tumor cellline specific to Fischer rats) into the hind legs using 25-gaugeneedles. Studies performed 14 to 17 days after implantation when tumorsreached approximately 1 cm diameter. Rats were anesthetized withketamine (10-15 mg/rat, intraperitoneally) before each procedure.

In tissue distribution studies, each animal was injected intravenouslywith 10-20 μCi of ^(99m)Tc-EC or ^(99m)Tc-EC-neomycin (n=3/time point).The injected mass of ^(99m)Tc-EC-neomycin was 200 μg per rat. At 0.5, 2and 4 hours following administration of the radiotracers, the rats weresacrificed and the selected tissues were excised, weighed and countedfor radioactivity. The biodistribution of tracer in each sample wascalculated as percentage of the injected dose per gram of tissue wetweight (% ID/g). Tumor/nontarget tissue count density ratios werecalculated from the corresponding % ID/g values. When compared to^(99m)Tc-EC (Table 4) and free technetium (Table 9), tumor-to tissueratios increased as a function of time in ^(99m)Tc-EC-neomycin group(Table 8).

Scintigraphic Imaging Studies

Scintigraphic images, using a gamma camera (Siemens Medical Systems,Inc., Hoffman Estates, Ill.) equipped with low-energy, parallel-holecollimator, were obtained 0.5, 2 and 4 hours after i.v. injection of 100μCi of each radiotracer. Compare to ^(99m)Tc-EC, high uptake in thetumors was observed (FIG. 37A). Preliminary clinical imaging studieswere conducted in a patient with breast cancer. The tumor was visualizedwell at 2 hours post-administration of ^(99m)Tc-EC-neomycin (FIG. 37B).TABLE 8 Biodistribution of ^(99m)Tc-EC-neomycin in Breast Tumor BearingRats 30 Min. 1 Hour 2 Hour 4 Hour Blood 0.463 ± 0.007 0.262 ± 0.0400.139 ± 0.016 0.085 ± 0.004 Lung 0.344 ± 0.011 0.202 ± 0.030 0.114 ±0.014 0.080 ± 0.003 Liver 0.337 ± 0.012 0.269 ± 0.013 0.221 ± 0.0200.195 ± 0.012 Stomach 0.279 ± 0.039 0.147 ± 0.001 0.061 ± 0.008 0.054 ±0.008 Spleen 0.159 ± 0.008 0.114 ± 0.013 0.095 ± 0.007 0.089 ± 0.003Kidney 8.391 ± 0.395 8.804 ± 0.817 8.356 ± 0.408 8.638 ± 0.251 Thyroid0.349 ± 0.008 0.202 ± 0.028 0.114 ± 0.007 0.086 ± 0.001 Muscle 0.093 ±0.001 0.049 ± 0.010 0.021 ± 0.006 0.010 ± 0.001 Intestine 0.159 ± 0.0040.093 ± 0.014 0.061 ± 0.004 0.266 ± 0.200 Urine 25.402 ± 8.621  21.786 ±2.690  0.224 ± 0.000 2.609 ± 2.377 Tumor 0.419 ± 0.023 0.279 ± 0.0420.166 ± 0.023 0.131 ± 0.002 Brain 0.022 ± 0.001 0.014 ± 0.003 0.010 ±0.001 0.007 ± 0.001 Heart 0.147 ± 0.009 0.081 ± 0.012 0.040 ± 0.0040.029 ± 0.002 Tumor/Blood 0.906 ± 0.039 1.070 ± 0.028 1.196 ± 0.0611.536 ± 0.029 Tumor/Muscle 4.512 ± 0.220 5.855 ± 0.458 8.364 ± 1.46912.706 ± 0.783  Tumor/Brain 19.495 ± 1.823  20.001 ± 0.890  17.515 ±2.035  20.255 ± 1.693 Values shown represent the mean ± standard deviation of data from 3animals.

TABLE 9 Biodistribution of ^(99m)Tc Pertechnetate in Breast TumorBearing Rats 30 Min. 2 Hour 4 Hour Blood 1.218 ± 0.328 0.666 ± 0.0660.715 ± 0.052 Lung 0.646 ± 0.291 0.632 ± 0.026 0.387 ± 0.024 Liver 0.541± 0.232 0.304 ± 0.026 0.501 ± 0.081 Spleen 0.331 ± 0.108 0.187 ± 0.0140.225 ± 0.017 Kidney 0.638 ± 0.197 0.489 ± 0.000 0.932 ± 0.029 Thyroid24.821 ± 5.181  11.907 ± 15.412 17.232 ± 5.002  Muscle 0.130 ± 0.0790.076 ± 0.002 0.063 ± 0.003 Intestine 0.153 ± 0.068 0.186 ± 0.007 0.344± 0.027 Tumor 0.591 ± 0.268 0.328 ± 0.016 0.423 ± 0.091 Brain 0.038 ±0.014 0.022 ± 0.002 0.031 ± 0.009 Heart 0.275 ± 0.089 0.145 ± 0.0150.166 ± 0.012 Tumor/Blood 0.472 ± 0.093 0.497 ± 0.073 0.597 ± 0.144Tumor/Muscle 4.788 ± 0.833 4.302 ± 0.093 6.689 ± 1.458 Tumor/Liver 1.084± 0.023 1.084 ± 0.115 0.865 ± 0.270Values shown represent the mean ± standard deviation of data from 3animals.In Vitro Cellular Uptake of ^(99m)Tc-EC-Drug Conjugates

To evaluate the cellular uptake of ^(99m)Tc-EC-drug conjugates, eachwell containing 80,000 cells (A549 lung cancer cell line) was added with2 μCi of ^(99m)Tc-EC-neomycin and ¹⁸F-FDG. After incubation at 0.5-4hours, the cells were washed with phosphate buffered saline 3 times andfollowed by trypsin to lose the cells. The cells were then counted by agamma counter. ^(99m)Tc-EC-neomycin showed highest uptake among thoseagents tested in human lung cancer cell line (FIG. 46).

Effect of Glucose on Cellular Uptake of ^(99m)Tc-EC-Neomycin and ¹⁸F-FDG

Neomycin is known to influence glucose absorption (Rogers et al., 1968;Fanciulli et al., 1994). Previous experiments have shown that^(99m)Tc-EC-neomycin has higher uptake than ¹⁸F-FDG in human lung cancercell line (A549). To determine if uptake of ^(99m)Tc-EC-neomycin ismediated via glucose-related mechanism, glucose (0.1 mg-2.0 mg) wasadded to each well containing either 50,000 (breast) cells or 80,000cells (lung) along with 2 μCi of ^(99m)Tc-EC-neomycin and ¹⁸F-FDG. Afterincubation, the cells were washed with phosphate buffered saline 3 timesand followed by trypsin to lose the cells. The cells were then countedby a gamma counter.

By adding glucose at the concentration of 0.1-2.0 mg/well, decreaseduptake of ^(99m)Tc-EC-neomycin in two lung cancer cell lines and onebreast cell line was observed. Similar results were observed in ¹⁸F-FDGgroups. ^(99m)Tc-EC (control) showed no uptake. The findings suggestthat the cellular uptake of ^(99m)Tc-EC-neomycin may be mediated viaglucose-related mechanism (FIGS. 47, 48A and 48B).

EXAMPLE 7 Tumor Metabolic Imaging with ^(99m)Tc-EC-Deoxyglucose

Synthesis of EC-Deoxyglucose (EC-DG)

Sodium hydroxide (1N, 1 ml) was added to a stirred solution of EC (110mg, 0.41 mmol) in water (5 ml). To this colorless solution, sulfo-NHS(241.6 mg, 1.12 mmol) and EDC (218.8 mg, 1.15 mmol) were added.D-Glucosamine hydrochloride salt (356.8 mg, 1.65 mmol) was then added.The mixture was stirred at room temperature for 24 hours. The mixturewas dialyzed for 48 hours using Spectra/POR molecular porous membranewith cut-off at 500 (Spectrum Medical Industries Inc., Houston, Tex.).After dialysis, the product was frozen dried using lyophilizer(Labconco, Kansas City, Mo.). The product in the salt form weighed 568.8mg. The synthetic scheme is shown in FIG. 59. The structure wasconfirmed by mass spectrometry (FIG. 60) and proton NMR (FIGS. 61 and62). Radiochemical purity of ^(99m)Tc-EC-DG was 100% as determined byradio-TLC (FIG. 63) and HPLC (FIGS. 64 and 65) analysis.

Hexokinase Assay

To determine if EC-DG mimics glucose phosphorylation, a hexokinase assaywas conducted. Using a ready made kit (Sigma Chemical Company), EC-DG,glucosamine and glucose (standard) were assayed at UV wavelength 340 nm.Glucose, EC-DG and glucosamine showed positive hexokinase assay (FIGS.66-68).

In Vitro Cellular Uptake Assay

In vitro cellular uptake assay was conducted by using a human lungcancer cell line (A549). Two μCi of ^(99m)Tc-EC-DG and ¹⁸F-FDG wereadded to wells containing 80,000 cells each. After incubation at 0.5-4hours, the cells were washed with phosphate buffered saline 3 times andfollowed by trypsin to lose the cells. The cells were then counted by agamma counter. The uptake of ^(99m)Tc-EC-DG was comparable to FDG (FIG.69).

Effect of d- and l-Glucose on Cellular Uptake of^(99m)Tc-EC-Deoxyglucose and ¹⁸F-FDG

To evaluate if the uptake of ^(99m)Tc-EC-deoxyglucose is mediated viad-glucose mechanism, d- and 1-glucose (1 mg and 2.0 mg) were added to,each well containing either breast or lung cancer cells (50,000/0.5ml/well), along with 2 μCi of ^(99m)Tc-EC-deoxyglucose and ¹⁸F-FDG.After 2 hours incubation, the cells were washed with phosphate bufferedsaline 3 times and followed by trypsin to lose the cells. The cells werecounted by a gamma counter.

By adding glucose at the concentration of 1-2.0 mg/well, a decreaseduptake of ^(99m)Tc-EC-deoxyglucose and ¹⁸F-FDG by d-glucose in breastand lung cancer cells was observed. However, there was no influence onboth agents by 1-glucose (FIG. 70-73). The findings suggest that thecellular uptake of ^(99m)Tc-EC-deoxyglucose is mediated via d-glucosemechanism.

Effect of EC-Deoxyglucose Loading on Blood Glucose Level in Normal Rats

Previous experiments have shown that cellular uptake of^(99m)Tc-EC-deoxyglucose is similar to FDG. For instance, the hexokinaseassay (glucose phosphorylation) was positive. The uptake of^(99m)Tc-EC-deoxyglucose is mediated via d-glucose mechanism. This studyis to determine whether blood glucose level could be induced by eitherFDG or EC-deoxyglucose and suppressed by insulin.

Normal healthy Fischer 344 rats (weight 145-155 g) were fastingovernight prior to the experiments. The concentration of glucosaminehydrochloride, FDG and EC-deoxyglucose prepared was 60% and 164%(mg/ml). The blood glucose level (mg/dl) was determined by a glucosemeter (Glucometer DEX, Bayer Corporation, Elkhart, Ind.). Prior to thestudy, the baseline of blood glucose level was obtained. Each rat(n=3/group) was administered 1.2 mmol/kg of glucosamine, FDG andEC-deoxyglucose. In a separate experiment, a group of rats wasadministered EC-deoxyglucose and FDG. Insulin (5 units) was administeredafter 30 minutes. Blood samples were collected from the tail vein every30 minutes up to 6 hours post-administration.

Blood glucose level was induced by bolus intravenous administration ofglucosamine, FDG and EC-deoxyglucose. This increased blood glucose levelcould be suppressed by co-administration of EC-deoxyglucose or FDG andinsulin (FIGS. 74 and 75).

Tissue Distribution Studies of ^(99m)Tc-EC-DG

For breast tumor-bearing animal model, female Fischer 344 rats (150±25g) (Harlan Sprague-Dawley, Indianapolis, Ind.) were innoculatedsubcutaneously with 0.1 ml of mammary tumor cells from the 13762 tumorcell line suspension (10⁶ cells/rat, a tumor cell line specific toFischer rats) into the hind legs using 25-gauge needles. Studies wereperformed 14 to 17 days after implantation when tumors reachedapproximately 1 cm diameter. Rats were anesthetized with ketamine (10-15mg/rat, intraperitoneally) before each procedure.

For lung tumor-bearing animal model, each athymic nude mouse (20-25 g)was innoculated subcutaneously with 0.1 ml of human lung tumor cellsfrom the A549 tumor cell line suspension (10⁶ cells/mouse) into the hindlegs using 25-gauge needles. Studies were performed 17 to 21 days afterimplantation when tumors reached approximately 0.6 cm diameter.

In tissue distribution studies, each animal was injected intravenouslywith 10-20 μCi (per rat) or 1-2 μCi (per mouse) of ^(99m)Tc-EC or^(99m)Tc-EC-DG (n=3/time point). The injected mass of ^(99m)Tc-EC-DG was1 mg per rat. At 0.5, 2 and 4 hours following administration of theradiotracers, the rodents were sacrificed and the selected tissues wereexcised, weighed and counted for radioactivity. The biodistribution oftracer in each sample was calculated as percentage of the injected doseper gram of tissue wet weight (% ID/g). Tumor/nontarget tissue countdensity ratios were calculated from the corresponding % ID/g values.When compared to ^(99m)Tc-EC (Table 4) and free technetium (Table 9),tumor-to tissue ratios increased as a function of time in ^(99m)Tc-EC-DGgroup (FIGS. 76-80).

Scintigraphic Imaging Studies

Scintigraphic images, using a gamma camera equipped with low-energy,parallel-hole collimator, were obtained 0.5, 2 and 4 hours after i.v.injection of 100 μCi of the radiotracer. The animal model used wasbreast tumor-bearing rats. Tumor could be visualized well when comparedto ^(99m)Tc-EC (control group) (FIG. 81). Preliminary clinical studieswere conducted in 5 patients (3 brain tumors and 2 lung diseases). Theimages were obtained at 1-2 hours post-administration. ^(99m)Tc-EC-DGwas able to differentiate benign versus malignant tumors. For instance,malignant astrocytoma showed high uptake (FIGS. 82A, 82B, 83A and 83B).Benign meningioma showed poor uptake compared to malignant meningioma(FIGS. 84A and B). Poor uptake was observed in patient with TB (FIG. 85Aand FIG. 85B), but high uptake was observed in lung tumor (FIG. 86A,FIG. 86B, and FIG. 86C).

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1-41. (canceled)
 42. A kit for preparing a radiopharmaceuticalpreparation, said kit comprising one or more sealed containers, and apredetermined quantity of a bis-aminoethanethiol (BAT)-targeting ligandconjugate composition in one or more of the sealed containiners.
 43. Thekit of claim 42, wherein the kit further comprises a reducing agent. 44.The kit of claim 42, wherein the targeting ligand is an anticanceragent, DNA topoisomerase inhibitor, antimetabolite, tumor marker, folatereceptor targeting ligand, tumor apoptotic cell targeting ligand, tumorhypoxia targeting ligand, DNA intercalator, receptor marker, peptide,organ ligand, antibiotic, antifungal, glutamate pentapeptide or an agentthat mimics glucose.
 45. The kit of claim 42, wherein the targetingligand is estradiol, topotecan, paclitaxel, raloxifen, etoposide,doxorubricin, mitomycin C, endostatin, annexin V, LHRH, octreotide, VIP,methotrexate or folic acid.
 46. The kit of claim 42, wherein thecomposition further comprises a linker between said bis-aminoethanethiol(BAT) and said targeting ligand.
 47. A reagent for preparing ascintigraphic imaging agent comprising a targeting ligand covalentlylinked to a bis-aminoethanethiol (BAT).
 48. The reagent of claim 47,wherein the bis-aminoethanethiol (BAT) is ethylenedicysteine.
 49. Thereagent of claim 47, wherein the targeting ligand is an anticanceragent, DNA topoisomerase inhibitor, antimetabolite, tumor marker, folatereceptor targeting ligand, tumor apoptotic cell targeting ligand, tumorhypoxia targeting ligand, DNA intercalator, receptor marker, peptide,organ specific ligand, antibiotic, antifungal, glutamate pentapeptide oran agent that mimics glucose.
 50. The reagent of claim 42, furthercomprising a linker between said targeting ligand and saidbis-aminoethanethiol (BAT).
 51. (canceled)
 52. The kit of claim 42,wherein the bis-aminoethanethiol (BAT)-targeting ligand isethylenedicysteine (EC).
 53. The kit of claim 42, further defined ascomprising a sealed containing comprising a reducing agent.
 54. The kitof claim 53, wherein the bis-aminoethanethiol (BAT)-targeting ligandconjugate composition and the reducing agent are comprised together in asealed container.
 55. The kit of claim 42, further defined as comprisinga sealed containing comprising a linker.
 56. The kit of claim 42,wherein the kit comprises a first sealed containing comprising thebis-aminoethanethiol (BAT)-targeting ligand conjugate composition and asecond sealed containing comprising a reducing agent.
 57. The kit ofclaim 42, wherein one or more of the sealed containers is furtherdefined as a bag.
 58. The kit of claim 43, wherein the reducing agent isa dithionite ion, a stannous ion or a ferrous ion.
 59. The kit of claim42, wherein the targeting ligand is an anticancer agent.
 60. The kit ofclaim 59, wherein the anticancer agent is methotrexate, doxorubicin,tamoxifen, paclitaxel, topotecan, LHRH, mitomycin C, etoposide tomudex,podophyllotoxin, mitoxantrone, camptothecin, colchicine, endostatin,fludarabin, gemcitabine, or tomudex.
 61. The kit of claim 42, whereinthe targeting ligand is an agent that mimics glucose.
 62. The kit ofclaim 61, wherein the agent that mimics glucose is glucosamine,deoxyglucose, neomycin, kanamycin, gentamicin, paromycin, amikacin,tobramycin, netilmicin, ribostamycin, sisomicin, micromicin,lividomycin, dibekacin, isepamicin, astromicin, or an aminoglycoside.63. The kit of claim 42, wherein the targeting ligand is a tumor marker.64. The kit of claim 64, wherein the tumor marker is PSA, ER, PR,CA-125, CA-199, CEA AFP, interferons, BRCA1, HER-2/neu, cytoxan, p53, orendostatin.
 65. The kit of claim 42, wherein the targeting ligand is aDNA topoisomerase inhibitor.
 66. The kit of claim 42, wherein thetargeting ligand is a folate receptor targeting ligand.
 67. The kit ofclaim 66, wherein the folate receptor targeting ligand is folate,methotrexate, or tomudex.
 68. The kit of claim 42, wherein the targetingligand is a tumor apoptotic cell targeting ligand or a tumor hypoxiatargeting ligand.
 69. The kit of claim 68, wherein the targeting ligandis annexin V, colchicine, nitroimidazole, mitomycin, or metronidazole.70. The kit of claim 42, wherein the targeting ligand is glutamatepentapeptide.
 71. The kit of claim 42, wherein the components of the kitare in a liquid form, frozen form, or dry form.
 72. The kit of claim 42,wherein the components of the kit are in a lyophilized form.
 73. The kitof claim 46, wherein the linker is a water soluble peptide, glutamicacid, polyglutamic acid, aspartic acid, polyaspartic acid,bromoethylacetate, ethylenediamine or lysine.
 74. The kit of claim 42,wherein the kit further comprises one or more pharmaceutical adjunctmaterials.
 75. The kit of claim 74, wherein the one or morepharmaceutical adjunct materials are selected from the group consistingof a pharmaceutically acceptable salt, a buffer, a preservative, andantioxidant, and a scavenger
 76. The kit of claim 75, wherein the kitcomprises an antioxidant.
 77. The kit of claim 76, wherein theantioxidant is vitamin C.
 78. The kit of claim 75, wherein the kitcomprises a scavenger.
 79. The kit of claim 78, wherein the scavenger isglucoheptonate or EDTA.
 80. The kit of claim 42, wherein the kit is ahome-made kit.
 81. The kit of claim 42, wherein the kit comprises EC,SnCl₂, Na₂IPO₄, ascorbic acid, and NaEDTA.
 82. The kit of claim 81,wherein the kit comprises lyophilized EC (3 mg), SnCl₂ (100 μg), Na₂IPO₄(13.5 mg), ascorbic acid (0.5 mg) and NaEDTA (0.5 mg) at pH
 10. 83. Thekit of claim 42, wherein the kit comprises EC-folate, SnCl₂, Na₂IPO₄,ascorbic acid, and NaEDTA.
 84. The kit of claim 83, wherein the kitcomprises lyophilized residue of EC-folate (3 mg), SnCl₂ (100 μg),Na₂IPO₄ (13.5 mg), ascorbic acid (0.5 mg), and NaEDTA (0.5 mg).
 85. Thekit of claim 42, wherein the kit comprises EC-MN or EC-NIM, SnCl₂,Na₂IPO₄, ascorbic acid, and NaEDTA.
 86. The kit of claim 85, wherein thekit comprises lyophilized residue of EC-MN or EC-NIM (3 mg), SnCl₂ (100μg), Na₂IPO₄ (13.5 mg), ascorbic acid (0.5 mg), and NaEDTA (0.5 mg).